{"Affiliation":[{"label":"Affiliation","value":"Medicine, Faculty of","attrs":{"lang":"en","ns":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","classmap":"vivo:EducationalProcess","property":"vivo:departmentOrSchool"},"iri":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","explain":"VIVO-ISF Ontology V1.6 Property; The department or school name within institution; Not intended to be an institution name."},{"label":"Affiliation","value":"Biochemistry and Molecular Biology, Department of","attrs":{"lang":"en","ns":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","classmap":"vivo:EducationalProcess","property":"vivo:departmentOrSchool"},"iri":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","explain":"VIVO-ISF Ontology V1.6 Property; The department or school name within institution; Not intended to be an institution name."}],"AggregatedSourceRepository":[{"label":"Aggregated Source Repository","value":"DSpace","attrs":{"lang":"en","ns":"http:\/\/www.europeana.eu\/schemas\/edm\/dataProvider","classmap":"ore:Aggregation","property":"edm:dataProvider"},"iri":"http:\/\/www.europeana.eu\/schemas\/edm\/dataProvider","explain":"A Europeana Data Model Property; The name or identifier of the organization who contributes data indirectly to an aggregation service (e.g. Europeana)"}],"Campus":[{"label":"Campus","value":"UBCV","attrs":{"lang":"en","ns":"https:\/\/open.library.ubc.ca\/terms#degreeCampus","classmap":"oc:ThesisDescription","property":"oc:degreeCampus"},"iri":"https:\/\/open.library.ubc.ca\/terms#degreeCampus","explain":"UBC Open Collections Metadata Components; Local Field; Identifies the name of the campus from which the graduate completed their degree."}],"Creator":[{"label":"Creator","value":"Rogers, Lindsay Deborah","attrs":{"lang":"en","ns":"http:\/\/purl.org\/dc\/terms\/creator","classmap":"dpla:SourceResource","property":"dcterms:creator"},"iri":"http:\/\/purl.org\/dc\/terms\/creator","explain":"A Dublin Core Terms Property; An entity primarily responsible for making the resource.; Examples of a Contributor include a person, an organization, or a service."}],"DateAvailable":[{"label":"Date Available","value":"2011-04-13T14:27:32Z","attrs":{"lang":"en","ns":"http:\/\/purl.org\/dc\/terms\/issued","classmap":"edm:WebResource","property":"dcterms:issued"},"iri":"http:\/\/purl.org\/dc\/terms\/issued","explain":"A Dublin Core Terms Property; Date of formal issuance (e.g., publication) of the resource."}],"DateIssued":[{"label":"Date Issued","value":"2011","attrs":{"lang":"en","ns":"http:\/\/purl.org\/dc\/terms\/issued","classmap":"oc:SourceResource","property":"dcterms:issued"},"iri":"http:\/\/purl.org\/dc\/terms\/issued","explain":"A Dublin Core Terms Property; Date of formal issuance (e.g., publication) of the resource."}],"Degree":[{"label":"Degree (Theses)","value":"Doctor of Philosophy - PhD","attrs":{"lang":"en","ns":"http:\/\/vivoweb.org\/ontology\/core#relatedDegree","classmap":"vivo:ThesisDegree","property":"vivo:relatedDegree"},"iri":"http:\/\/vivoweb.org\/ontology\/core#relatedDegree","explain":"VIVO-ISF Ontology V1.6 Property; The thesis degree; Extended Property specified by UBC, as per https:\/\/wiki.duraspace.org\/display\/VIVO\/Ontology+Editor%27s+Guide"}],"DegreeGrantor":[{"label":"Degree Grantor","value":"University of British Columbia","attrs":{"lang":"en","ns":"https:\/\/open.library.ubc.ca\/terms#degreeGrantor","classmap":"oc:ThesisDescription","property":"oc:degreeGrantor"},"iri":"https:\/\/open.library.ubc.ca\/terms#degreeGrantor","explain":"UBC Open Collections Metadata Components; Local Field; Indicates the institution where thesis was granted."}],"Description":[{"label":"Description","value":"Salmonella enterica is an intracellular bacterium causing gastroenteritis and typhoid fever. Virulence is achieved by two type III secretion systems (T3SS) encoded on Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) that translocate effector proteins into host cells where they mimic or block host protein function. Effectors translocated by T3SS-1 facilitate internalization of the bacteria into the Salmonella-containing vacuole (SCV), actively stimulate intracellular signaling cascades, and regulate trafficking of the SCV to avoid degradation. A T3SS-1 effector, SopB has been shown to regulate a vast array of host processes important for pathogenesis, but only a few host proteins have been identified as targets of this effector. Here quantitative mass spectrometry-based proteomics and bioinformatics techniques have been employed to identify novel host targets of SopB. Quantitative immunoprecipitation experiments identified Cell division control protein 42 (Cdc42) as a direct SopB binding partner, and the binding site within SopB was localized to residues 117-168. SopB and active Cdc42 were shown to colocalize at membrane ruffles on the host cell surface, and two SopB monoubiquitylation sites were identified. To globally analyze host protein phosphorylation events regulated by SopB, a phosphoproteomics method employing heat and chaotropic denaturation for phosphatase inactivation, peptide fractionation by in-solution isoelectric focusing, and phosphopeptide enrichment by metal oxide chromatography was developed. Quantitative analysis of host protein phosphorylation during the initial 20 minutes post Salmonella infection identified >9000 phosphorylation sites, >2000 of which were dynamic. Signaling cascades downstream of T3SS-1 were compared to those induced by growth factor simulation, revealing stark differences between these signaling mechanisms. Kinase prediction upstream of dynamic phosphosites revealed protein kinases B and C as master host regulators during Salmonella infection, and phosphorylation dynamics following wild type versus \u0394sopB infection were compared to identify novel host targets of SopB. This work has greatly improved our understanding of SopB\u2019s activity within host cells. It has also provided the first global view of host protein phosphorylation dynamics during bacterial infection, and developed several techniques that can be widely applied within the field of pathogen-host interactions.","attrs":{"lang":"en","ns":"http:\/\/purl.org\/dc\/terms\/description","classmap":"dpla:SourceResource","property":"dcterms:description"},"iri":"http:\/\/purl.org\/dc\/terms\/description","explain":"A Dublin Core Terms Property; An account of the resource.; Description may include but is not limited to: an abstract, a table of contents, a graphical representation, or a free-text account of the resource."}],"DigitalResourceOriginalRecord":[{"label":"Digital Resource Original Record","value":"https:\/\/circle.library.ubc.ca\/rest\/handle\/2429\/33597?expand=metadata","attrs":{"lang":"en","ns":"http:\/\/www.europeana.eu\/schemas\/edm\/aggregatedCHO","classmap":"ore:Aggregation","property":"edm:aggregatedCHO"},"iri":"http:\/\/www.europeana.eu\/schemas\/edm\/aggregatedCHO","explain":"A Europeana Data Model Property; The identifier of the source object, e.g. the Mona Lisa itself. This could be a full linked open date URI or an internal identifier"}],"FullText":[{"label":"Full Text","value":"PROTEOMIC ANALYSIS OF SALMONELLA-HOST INTERACTIONS REVEALS NOVEL HOST TARGETS OF SOPB by Lindsay Deborah Rogers B.Sc., Queen\u2019s University, 2005 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2011 \u00a9 Lindsay Deborah Rogers, 2011 \t\r \u00a0 ii\t\r \u00a0 Abstract\t\r \u00a0 Salmonella\t\r \u00a0enterica\t\r \u00a0is\t\r \u00a0an\t\r \u00a0intracellular\t\r \u00a0bacterium\t\r \u00a0causing\t\r \u00a0gastroenteritis\t\r \u00a0and\t\r \u00a0typhoid\t\r \u00a0fever.\t\r \u00a0Virulence\t\r \u00a0is\t\r \u00a0achieved\t\r \u00a0by\t\r \u00a0two\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0systems\t\r \u00a0(T3SS)\t\r \u00a0encoded\t\r \u00a0on\t\r \u00a0Salmonella\t\r \u00a0pathogenicity\t\r \u00a0islands\t\r \u00a01\t\r \u00a0and\t\r \u00a02\t\r \u00a0(SPI-\u00ad\u20101\t\r \u00a0and\t\r \u00a0SPI-\u00ad\u20102)\t\r \u00a0that\t\r \u00a0translocate\t\r \u00a0effector\t\r \u00a0proteins\t\r \u00a0into\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0where\t\r \u00a0they\t\r \u00a0mimic\t\r \u00a0or\t\r \u00a0block\t\r \u00a0host\t\r \u00a0protein\t\r \u00a0function.\t\r \u00a0Effectors\t\r \u00a0translocated\t\r \u00a0by\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0facilitate\t\r \u00a0internalization\t\r \u00a0of\t\r \u00a0the\t\r \u00a0bacteria\t\r \u00a0into\t\r \u00a0the\t\r \u00a0Salmonella-\u00ad\u2010containing\t\r \u00a0vacuole\t\r \u00a0(SCV),\t\r \u00a0actively\t\r \u00a0stimulate\t\r \u00a0intracellular\t\r \u00a0signaling\t\r \u00a0cascades,\t\r \u00a0and\t\r \u00a0regulate\t\r \u00a0trafficking\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV\t\r \u00a0to\t\r \u00a0avoid\t\r \u00a0degradation.\t\r \u00a0A\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0effector,\t\r \u00a0SopB\t\r \u00a0has\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0regulate\t\r \u00a0a\t\r \u00a0vast\t\r \u00a0array\t\r \u00a0of\t\r \u00a0host\t\r \u00a0processes\t\r \u00a0important\t\r \u00a0for\t\r \u00a0pathogenesis,\t\r \u00a0but\t\r \u00a0only\t\r \u00a0a\t\r \u00a0few\t\r \u00a0host\t\r \u00a0proteins\t\r \u00a0have\t\r \u00a0been\t\r \u00a0identified\t\r \u00a0as\t\r \u00a0targets\t\r \u00a0of\t\r \u00a0this\t\r \u00a0effector.\t\r \u00a0Here\t\r \u00a0quantitative\t\r \u00a0mass\t\r \u00a0spectrometry-\u00ad\u2010based\t\r \u00a0proteomics\t\r \u00a0and\t\r \u00a0bioinformatics\t\r \u00a0techniques\t\r \u00a0have\t\r \u00a0been\t\r \u00a0employed\t\r \u00a0to\t\r \u00a0identify\t\r \u00a0novel\t\r \u00a0host\t\r \u00a0targets\t\r \u00a0of\t\r \u00a0SopB.\t\r \u00a0Quantitative\t\r \u00a0immunoprecipitation\t\r \u00a0experiments\t\r \u00a0identified\t\r \u00a0Cell\t\r \u00a0division\t\r \u00a0control\t\r \u00a0protein\t\r \u00a042\t\r \u00a0(Cdc42)\t\r \u00a0as\t\r \u00a0a\t\r \u00a0direct\t\r \u00a0SopB\t\r \u00a0binding\t\r \u00a0partner,\t\r \u00a0and\t\r \u00a0the\t\r \u00a0binding\t\r \u00a0site\t\r \u00a0within\t\r \u00a0SopB\t\r \u00a0was\t\r \u00a0localized\t\r \u00a0to\t\r \u00a0residues\t\r \u00a0117-\u00ad\u2010168.\t\r \u00a0SopB\t\r \u00a0and\t\r \u00a0active\t\r \u00a0Cdc42\t\r \u00a0were\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0colocalize\t\r \u00a0at\t\r \u00a0membrane\t\r \u00a0ruffles\t\r \u00a0on\t\r \u00a0the\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0surface,\t\r \u00a0and\t\r \u00a0two\t\r \u00a0SopB\t\r \u00a0monoubiquitylation\t\r \u00a0sites\t\r \u00a0were\t\r \u00a0identified.\t\r \u00a0To\t\r \u00a0globally\t\r \u00a0analyze\t\r \u00a0host\t\r \u00a0protein\t\r \u00a0phosphorylation\t\r \u00a0events\t\r \u00a0regulated\t\r \u00a0by\t\r \u00a0SopB,\t\r \u00a0a\t\r \u00a0phosphoproteomics\t\r \u00a0method\t\r \u00a0employing\t\r \u00a0heat\t\r \u00a0and\t\r \u00a0chaotropic\t\r \u00a0denaturation\t\r \u00a0for\t\r \u00a0phosphatase\t\r \u00a0inactivation,\t\r \u00a0peptide\t\r \u00a0fractionation\t\r \u00a0by\t\r \u00a0in-\u00ad\u2010solution\t\r \u00a0isoelectric\t\r \u00a0focusing,\t\r \u00a0and\t\r \u00a0phosphopeptide\t\r \u00a0enrichment\t\r \u00a0by\t\r \u00a0metal\t\r \u00a0oxide\t\r \u00a0chromatography\t\r \u00a0was\t\r \u00a0developed.\t\r \u00a0Quantitative\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0host\t\r \u00a0protein\t\r \u00a0phosphorylation\t\r \u00a0during\t\r \u00a0the\t\r \u00a0initial\t\r \u00a020\t\r \u00a0minutes\t\r \u00a0post\t\r \u00a0Salmonella\t\r \u00a0infection\t\r \u00a0identified\t\r \u00a0>9000\t\r \u00a0phosphorylation\t\r \u00a0sites,\t\r \u00a0>2000\t\r \u00a0of\t\r \u00a0which\t\r \u00a0were\t\r \u00a0dynamic.\t\r \u00a0Signaling\t\r \u00a0cascades\t\r \u00a0downstream\t\r \u00a0of\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0were\t\r \u00a0compared\t\r \u00a0 \t\r \u00a0 iii\t\r \u00a0 to\t\r \u00a0those\t\r \u00a0induced\t\r \u00a0by\t\r \u00a0growth\t\r \u00a0factor\t\r \u00a0simulation,\t\r \u00a0revealing\t\r \u00a0stark\t\r \u00a0differences\t\r \u00a0between\t\r \u00a0these\t\r \u00a0signaling\t\r \u00a0mechanisms.\t\r \u00a0Kinase\t\r \u00a0prediction\t\r \u00a0upstream\t\r \u00a0of\t\r \u00a0dynamic\t\r \u00a0phosphosites\t\r \u00a0revealed\t\r \u00a0protein\t\r \u00a0kinases\t\r \u00a0B\t\r \u00a0and\t\r \u00a0C\t\r \u00a0as\t\r \u00a0master\t\r \u00a0host\t\r \u00a0regulators\t\r \u00a0during\t\r \u00a0Salmonella\t\r \u00a0infection,\t\r \u00a0and\t\r \u00a0phosphorylation\t\r \u00a0dynamics\t\r \u00a0following\t\r \u00a0wild\t\r \u00a0type\t\r \u00a0versus\t\r \u00a0\u0394sopB\t\r \u00a0infection\t\r \u00a0were\t\r \u00a0compared\t\r \u00a0to\t\r \u00a0identify\t\r \u00a0novel\t\r \u00a0host\t\r \u00a0targets\t\r \u00a0of\t\r \u00a0SopB.\t\r \u00a0This\t\r \u00a0work\t\r \u00a0has\t\r \u00a0greatly\t\r \u00a0improved\t\r \u00a0our\t\r \u00a0understanding\t\r \u00a0of\t\r \u00a0SopB\u2019s\t\r \u00a0activity\t\r \u00a0within\t\r \u00a0host\t\r \u00a0cells.\t\r \u00a0It\t\r \u00a0has\t\r \u00a0also\t\r \u00a0provided\t\r \u00a0the\t\r \u00a0first\t\r \u00a0global\t\r \u00a0view\t\r \u00a0of\t\r \u00a0host\t\r \u00a0protein\t\r \u00a0phosphorylation\t\r \u00a0dynamics\t\r \u00a0during\t\r \u00a0bacterial\t\r \u00a0infection,\t\r \u00a0and\t\r \u00a0developed\t\r \u00a0several\t\r \u00a0techniques\t\r \u00a0that\t\r \u00a0can\t\r \u00a0be\t\r \u00a0widely\t\r \u00a0applied\t\r \u00a0within\t\r \u00a0the\t\r \u00a0field\t\r \u00a0of\t\r \u00a0pathogen-\u00ad\u2010host\t\r \u00a0interactions.\t\r \u00a0 \t\r \u00a0 iv\t\r \u00a0 Preface\t\r \u00a0 A\t\r \u00a0version\t\r \u00a0of\t\r \u00a0section\t\r \u00a01.3\t\r \u00a0has\t\r \u00a0been\t\r \u00a0published.\t\r \u00a0Rogers,\t\r \u00a0L.D.\t\r \u00a0and\t\r \u00a0Foster,\t\r \u00a0L.J.\t\r \u00a0(2009)\t\r \u00a0Phosphoproteomics\t\r \u00a0\u2013\t\r \u00a0finally\t\r \u00a0fulfilling\t\r \u00a0the\t\r \u00a0promise?\t\r \u00a0Mol\t\r 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\u00a0and\t\r \u00a0Foster,\t\r \u00a0L.J.\t\r \u00a0(2008)\t\r \u00a0Identification\t\r \u00a0of\t\r \u00a0cognate\t\r \u00a0host\t\r \u00a0targets\t\r \u00a0and\t\r \u00a0specific\t\r \u00a0ubiquitylation\t\r \u00a0sites\t\r \u00a0on\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0effector\t\r \u00a0SopB\/SigD.\t\r \u00a0J\t\r \u00a0Proteomics.\t\r \u00a071(1):97-\u00ad\u2010108.\t\r \u00a0I\t\r \u00a0was\t\r \u00a0the\t\r \u00a0primary\t\r \u00a0researcher\t\r \u00a0involved\t\r \u00a0in\t\r \u00a0this\t\r \u00a0project.\t\r \u00a0I,\t\r \u00a0along\t\r \u00a0with\t\r \u00a0ARK,\t\r \u00a0conducted\t\r \u00a0the\t\r \u00a0SILAC\t\r \u00a0immunoprecipitation\t\r \u00a0experiments.\t\r \u00a0I\t\r \u00a0conducted\t\r \u00a0the\t\r \u00a0microscopy\t\r \u00a0experiments,\t\r \u00a0experiments\t\r \u00a0assaying\t\r \u00a0Cdc42\t\r \u00a0activity,\t\r \u00a0and\t\r \u00a0experiments\t\r \u00a0localizing\t\r \u00a0the\t\r \u00a0Cdc42\t\r \u00a0binding\t\r 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\u00a0by\t\r \u00a0permission\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Royal\t\r \u00a0Society\t\r \u00a0of\t\r \u00a0Chemistry\t\r \u00a0http:\/\/pubs.rsc.org\/en\/Content\/ArticleLanding\/2010\/MB\/b915986j.\t\r \u00a0\t\r \u00a0The\t\r \u00a0work\t\r \u00a0in\t\r \u00a0chapter\t\r \u00a04\t\r \u00a0has\t\r \u00a0been\t\r \u00a0submitted\t\r \u00a0for\t\r \u00a0publication.\t\r \u00a0Rogers,\t\r \u00a0L.D.,\t\r \u00a0Fang,\t\r \u00a0Y.,\t\r \u00a0Brown,\t\r \u00a0N.F.,\t\r \u00a0Pelech,\t\r \u00a0S.\t\r \u00a0and\t\r \u00a0Foster,\t\r \u00a0L.J.\t\r \u00a0SopB\t\r \u00a0is\t\r \u00a0a\t\r \u00a0master\t\r \u00a0regulator\t\r \u00a0of\t\r \u00a0host\t\r \u00a0signaling\t\r \u00a0cascades\t\r \u00a0during\t\r \u00a0Salmonella\t\r \u00a0infection.\t\r \u00a0I\t\r \u00a0was\t\r \u00a0the\t\r \u00a0primary\t\r \u00a0researcher\t\r \u00a0involved\t\r \u00a0in\t\r \u00a0this\t\r \u00a0project.\t\r \u00a0I\t\r \u00a0conducted\t\r \u00a0the\t\r \u00a0phosphoproteomics\t\r \u00a0experiments\t\r 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\u00a0supervisor\t\r \u00a0LJF.\t\r \u00a0 \t\r \u00a0 vi\t\r \u00a0 Table\t\r \u00a0of\t\r \u00a0Contents\t\r \u00a0 Abstract\t\r \u00a0..........................................................................................................................................................\t\r \u00a0ii\t\r \u00a0Preface\t\r \u00a0...........................................................................................................................................................\t\r \u00a0iv\t\r \u00a0Table\t\r \u00a0of\t\r \u00a0Contents\t\r \u00a0......................................................................................................................................\t\r \u00a0vi\t\r \u00a0List\t\r \u00a0of\t\r \u00a0Tables\t\r \u00a0.............................................................................................................................................\t\r \u00a0xii\t\r \u00a0List\t\r \u00a0of\t\r \u00a0Figures\t\r \u00a0..........................................................................................................................................\t\r \u00a0xiii\t\r \u00a0List\t\r \u00a0of\t\r \u00a0Abbreviations\t\r \u00a0...............................................................................................................................\t\r \u00a0xv\t\r \u00a0Acknowledgements\t\r \u00a0..............................................................................................................................\t\r \u00a0xxii\t\r \u00a0Dedication\t\r \u00a0...............................................................................................................................................\t\r \u00a0xxiii\t\r \u00a01\t\r \u00a0Introduction\t\r \u00a0..............................................................................................................................................\t\r \u00a01\t\r \u00a01.1\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0.......................................................................................................................\t\r \u00a01\t\r \u00a01.1.1\t\r \u00a0Salmonella\t\r \u00a0phylogeny\t\r \u00a0.........................................................................................................\t\r \u00a01\t\r \u00a01.1.2\t\r \u00a0Salmonella\t\r \u00a0and\t\r \u00a0human\t\r \u00a0health\t\r \u00a0..........................................................................................\t\r \u00a01\t\r \u00a01.1.3\t\r \u00a0Salmonella\t\r \u00a0pathogenesis\t\r \u00a0....................................................................................................\t\r \u00a03\t\r \u00a01.1.3.1\t\r \u00a0Salmonella\t\r \u00a0pathogenesis\t\r \u00a0within\t\r \u00a0host\t\r \u00a0organisms\t\r \u00a0............................................\t\r \u00a03\t\r \u00a01.1.3.2\t\r \u00a0Cellular\t\r \u00a0aspects\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0pathogenesis\t\r \u00a0....................................................\t\r \u00a04\t\r \u00a01.1.3.2.1\t\r \u00a0Salmonella-\u00ad\u2010induced\t\r \u00a0signaling\t\r \u00a0cascades\t\r \u00a0......................................................\t\r \u00a04\t\r \u00a01.1.3.2.2\t\r \u00a0Internalization\t\r \u00a0into\t\r \u00a0the\t\r \u00a0Salmonella-\u00ad\u2010containing\t\r \u00a0vacuole\t\r \u00a0.....................\t\r \u00a05\t\r \u00a01.1.3.2.3\t\r \u00a0Maturation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV\t\r \u00a0........................................................................................\t\r \u00a06\t\r \u00a01.1.3.2.4\t\r \u00a0Salmonella\u2019s\t\r \u00a0replicative\t\r \u00a0niche\t\r \u00a0.........................................................................\t\r \u00a08\t\r \u00a01.1.4\t\r \u00a0Genetics\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0virulence\t\r \u00a0.................................................................................\t\r \u00a010\t\r \u00a01.1.5\t\r \u00a0Salmonella-\u00ad\u2010pathogenicity\t\r \u00a0island\t\r \u00a01\t\r \u00a0..............................................................................\t\r \u00a012\t\r \u00a01.1.5.1\t\r \u00a0Regulation\t\r \u00a0of\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0...................................................................................................\t\r \u00a012\t\r \u00a0 \t\r \u00a0 vii\t\r \u00a0 1.1.5.2\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0apparatus\t\r \u00a0......................................................................................................\t\r \u00a013\t\r \u00a01.1.5.3\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0translocons\t\r \u00a0.......................................................................................................\t\r \u00a015\t\r \u00a01.1.5.4\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0effectors\t\r 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\u00a0....................................................................\t\r \u00a023\t\r \u00a01.1.5.5\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0chaperones\t\r \u00a0...................................................................................................\t\r \u00a024\t\r \u00a01.1.6\t\r \u00a0Salmonella-\u00ad\u2010pathogenicity\t\r \u00a0island\t\r \u00a02\t\r \u00a0..............................................................................\t\r \u00a025\t\r \u00a01.1.6.1\t\r \u00a0Regulation\t\r \u00a0of\t\r \u00a0SPI-\u00ad\u20102\t\r \u00a0...................................................................................................\t\r \u00a025\t\r \u00a01.1.6.2\t\r \u00a0T3SS-\u00ad\u20102\t\r \u00a0apparatus\t\r \u00a0......................................................................................................\t\r \u00a025\t\r \u00a01.1.6.3\t\r \u00a0T3SS-\u00ad\u20102\t\r \u00a0translocons\t\r 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\u00a0.....................................................................................................................................\t\r \u00a032\t\r \u00a01.2.3\t\r \u00a0LTQ-\u00ad\u2010Orbitrap\t\r \u00a0........................................................................................................................\t\r \u00a034\t\r \u00a01.2.4\t\r \u00a0Peptide\t\r \u00a0and\t\r \u00a0protein\t\r \u00a0identification\t\r \u00a0..............................................................................\t\r \u00a036\t\r \u00a01.2.5\t\r \u00a0Quantitation\t\r \u00a0..........................................................................................................................\t\r \u00a038\t\r \u00a01.2.6\t\r \u00a0Pre-\u00ad\u2010fractionation\t\r \u00a0and\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0PTMs\t\r \u00a0...................................................................\t\r \u00a041\t\r \u00a01.3\t\r \u00a0Phosphoproteomics\t\r \u00a0...................................................................................................................\t\r \u00a043\t\r \u00a0 \t\r \u00a0 viii\t\r \u00a0 1.3.1\t\r \u00a0History\t\r \u00a0and\t\r \u00a0current\t\r \u00a0potential\t\r \u00a0.......................................................................................\t\r \u00a043\t\r \u00a01.3.2\t\r \u00a0Lysis\t\r \u00a0methods\t\r \u00a0and\t\r \u00a0low\t\r \u00a0stoichiometry\t\r \u00a0of\t\r \u00a0phosphorylated\t\r \u00a0proteins\t\r \u00a0............\t\r \u00a044\t\r \u00a01.3.3\t\r \u00a0Phosphopeptide\t\r \u00a0pre-\u00ad\u2010fractionation\t\r \u00a0.............................................................................\t\r \u00a045\t\r \u00a01.3.4\t\r \u00a0Phosphopeptide\t\r \u00a0enrichment\t\r \u00a0.........................................................................................\t\r \u00a047\t\r \u00a01.3.5\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0methods\t\r \u00a0for\t\r \u00a0phosphoproteomics\t\r \u00a0................................................................\t\r \u00a050\t\r \u00a01.3.6\t\r \u00a0Phosphopeptide\t\r \u00a0and\t\r \u00a0phosphoprotein\t\r \u00a0databases\t\r \u00a0.................................................\t\r \u00a053\t\r \u00a01.4\t\r \u00a0Salmonella\t\r \u00a0and\t\r \u00a0proteomic\t\r \u00a0research\t\r \u00a0...................................................................................\t\r \u00a053\t\r \u00a01.5\t\r \u00a0Research\t\r \u00a0aims\t\r \u00a0and\t\r \u00a0hypothesis\t\r \u00a0..............................................................................................\t\r \u00a054\t\r \u00a02\t\r \u00a0Identification\t\r \u00a0of\t\r \u00a0cognate\t\r \u00a0host\t\r \u00a0targets\t\r \u00a0and\t\r \u00a0specific\t\r \u00a0ubiquitylation\t\r \u00a0sites\t\r \u00a0on\t\r \u00a0the\t\r \u00a0 Salmonella\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0effector\t\r \u00a0SopB\/SigD\t\r \u00a0.............................................................................................\t\r \u00a056\t\r \u00a02.1\t\r \u00a0Introduction\t\r \u00a0..................................................................................................................................\t\r \u00a056\t\r \u00a02.2\t\r \u00a0Experimental\t\r \u00a0procedures\t\r \u00a0........................................................................................................\t\r \u00a058\t\r \u00a02.2.1\t\r \u00a0Plasmid\t\r \u00a0sources\t\r \u00a0and\t\r \u00a0construction\t\r \u00a0..............................................................................\t\r \u00a058\t\r \u00a02.2.2\t\r \u00a0Preparation\t\r \u00a0of\t\r \u00a0GST-\u00ad\u2010SopB\t\r \u00a0protein\t\r \u00a0................................................................................\t\r \u00a060\t\r \u00a02.2.3\t\r \u00a0Cell\t\r \u00a0culture\t\r \u00a0and\t\r \u00a0transfections\t\r \u00a0.......................................................................................\t\r \u00a061\t\r \u00a02.2.4\t\r \u00a0SILAC\t\r \u00a02HA-\u00ad\u2010SopB\t\r \u00a0immunoprecipitations\t\r \u00a0.................................................................\t\r \u00a062\t\r \u00a02.2.5\t\r \u00a0GST-\u00ad\u2010SopB\t\r \u00a0pull\t\r \u00a0downs\t\r \u00a0.......................................................................................................\t\r \u00a063\t\r \u00a02.2.6\t\r \u00a0Mass\t\r \u00a0spectrometry\t\r \u00a0............................................................................................................\t\r \u00a064\t\r \u00a02.2.7\t\r \u00a0SopB\t\r \u00a0immunoprecipitation\t\r \u00a0............................................................................................\t\r \u00a065\t\r \u00a02.2.8\t\r \u00a0Immunoflourescence\t\r \u00a0........................................................................................................\t\r \u00a066\t\r \u00a02.2.9\t\r \u00a0Myc-\u00ad\u2010Cdc42\t\r \u00a0immunoprecipitations\t\r \u00a0.............................................................................\t\r \u00a067\t\r \u00a02.2.10\t\r \u00a0Cdc42\t\r \u00a0activity\t\r \u00a0assays\t\r \u00a0.....................................................................................................\t\r \u00a067\t\r \u00a02.3\t\r \u00a0Results\t\r \u00a0.............................................................................................................................................\t\r \u00a068\t\r \u00a0 \t\r \u00a0 ix\t\r \u00a0 2.3.1\t\r \u00a0Cdc42\t\r \u00a0is\t\r \u00a0the\t\r \u00a0host\t\r \u00a0protein\t\r \u00a0target\t\r \u00a0for\t\r \u00a0SopB\t\r \u00a0...............................................................\t\r \u00a068\t\r \u00a02.3.2\t\r \u00a0Confirmation\t\r \u00a0of\t\r \u00a0SopB-\u00ad\u2010Cdc42\t\r \u00a0interaction\t\r \u00a0.................................................................\t\r \u00a072\t\r \u00a02.3.3\t\r \u00a0Cdc42\t\r \u00a0is\t\r \u00a0a\t\r \u00a0host\t\r \u00a0target\t\r \u00a0of\t\r \u00a0bacterially\t\r \u00a0delivered\t\r \u00a0SopB\t\r \u00a0..........................................\t\r \u00a073\t\r \u00a02.3.4\t\r \u00a0Colocalization\t\r \u00a0of\t\r \u00a0Cdc42\t\r \u00a0and\t\r \u00a0truncated\t\r \u00a0SopB\t\r \u00a0proteins\t\r \u00a0.......................................\t\r \u00a075\t\r \u00a02.3.5\t\r \u00a0Cdc42\t\r \u00a0binds\t\r \u00a0SopB\t\r \u00a0between\t\r \u00a0resides\t\r \u00a0117-\u00ad\u2010168\t\r \u00a0........................................................\t\r \u00a078\t\r \u00a02.3.6\t\r \u00a0SopB\t\r \u00a0binding\t\r \u00a0does\t\r \u00a0not\t\r \u00a0activate\t\r \u00a0Cdc42\t\r \u00a0......................................................................\t\r \u00a079\t\r \u00a02.3.7\t\r \u00a0Identification\t\r \u00a0of\t\r \u00a0SopB\t\r \u00a0ubiquitylation\t\r \u00a0sites\t\r \u00a0..............................................................\t\r \u00a080\t\r \u00a02.4\t\r \u00a0Discussion\t\r \u00a0......................................................................................................................................\t\r \u00a082\t\r \u00a03\t\r \u00a0An\t\r \u00a0integrated\t\r \u00a0global\t\r \u00a0strategy\t\r \u00a0for\t\r \u00a0cell\t\r \u00a0lysis,\t\r \u00a0fractionation,\t\r \u00a0enrichment\t\r \u00a0and\t\r \u00a0mass\t\r \u00a0spectrometric\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0phosphorylated\t\r \u00a0peptides\t\r \u00a0...............................................................\t\r \u00a086\t\r \u00a03.1\t\r \u00a0Introduction\t\r \u00a0..................................................................................................................................\t\r \u00a086\t\r \u00a03.2\t\r \u00a0Experimental\t\r \u00a0procedures\t\r \u00a0........................................................................................................\t\r \u00a090\t\r \u00a03.2.1\t\r \u00a0Cell\t\r \u00a0culture\t\r \u00a0and\t\r \u00a0lysis\t\r \u00a0.........................................................................................................\t\r \u00a090\t\r \u00a03.2.2\t\r \u00a0Phosphate\t\r \u00a0assay\t\r \u00a0.................................................................................................................\t\r \u00a090\t\r \u00a03.2.3\t\r \u00a0Tryptic\t\r \u00a0digest\t\r \u00a0and\t\r \u00a0desalting\t\r \u00a0..........................................................................................\t\r \u00a092\t\r \u00a03.2.4\t\r \u00a0In-\u00ad\u2010solution\t\r \u00a0isoelectric\t\r \u00a0focusing\t\r \u00a0....................................................................................\t\r \u00a092\t\r \u00a03.2.5\t\r \u00a0Phosphopeptide\t\r \u00a0enrichment\t\r \u00a0.........................................................................................\t\r \u00a093\t\r \u00a03.2.6\t\r \u00a0Testing\t\r \u00a0interference\t\r \u00a0of\t\r \u00a0phosphopeptide\t\r \u00a0enrichment\t\r \u00a0by\t\r \u00a0phosphatase\t\r \u00a0inhibitors\t\r \u00a0...........................................................................................................................................\t\r \u00a094\t\r \u00a03.2.7\t\r \u00a0Mass\t\r \u00a0spectrometry\t\r \u00a0............................................................................................................\t\r \u00a094\t\r \u00a03.3\t\r \u00a0Results\t\r \u00a0.............................................................................................................................................\t\r \u00a095\t\r \u00a03.3.1\t\r \u00a0Phosphatase\t\r \u00a0inhibitors\t\r \u00a0versus\t\r \u00a0heat\/chaotropic\t\r \u00a0denaturation\t\r \u00a0for\t\r \u00a0minimizing\t\r \u00a0phosphatase\t\r \u00a0activity\t\r \u00a0...........................................................................................\t\r \u00a095\t\r \u00a0 \t\r \u00a0 x\t\r \u00a0 3.3.2\t\r \u00a0Phosphopeptide\t\r \u00a0prefractionation\t\r \u00a0using\t\r \u00a0solution-\u00ad\u2010based\t\r \u00a0isoelectric\t\r \u00a0focusing\t\r \u00a0..............................................................................................................................................\t\r \u00a099\t\r \u00a03.3.3\t\r \u00a0Binding\t\r \u00a0capacity\t\r \u00a0of\t\r \u00a0TiO2\t\r \u00a0for\t\r \u00a0phosphopeptides\t\r \u00a0...................................................\t\r \u00a0100\t\r \u00a03.3.4\t\r \u00a0Analysis\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0by\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0...............................................................\t\r \u00a0101\t\r \u00a03.4\t\r \u00a0Discussion\t\r \u00a0...................................................................................................................................\t\r \u00a0104\t\r \u00a04\t\r \u00a0SopB\t\r \u00a0is\t\r \u00a0a\t\r \u00a0master\t\r \u00a0regulator\t\r \u00a0of\t\r \u00a0host\t\r \u00a0signaling\t\r \u00a0cascades\t\r \u00a0during\t\r \u00a0Salmonella\t\r \u00a0infection\t\r \u00a0......................................................................................................................................................................\t\r \u00a0108\t\r \u00a04.1\t\r \u00a0Introduction\t\r \u00a0...............................................................................................................................\t\r \u00a0108\t\r \u00a04.2\t\r \u00a0Experimental\t\r \u00a0procedures\t\r \u00a0.....................................................................................................\t\r \u00a0110\t\r \u00a04.2.1\t\r \u00a0Cell\t\r \u00a0culture\t\r \u00a0and\t\r \u00a0Salmonella\t\r \u00a0infection\t\r \u00a0.....................................................................\t\r \u00a0110\t\r \u00a04.2.2\t\r \u00a0Sample\t\r \u00a0generation\t\r \u00a0for\t\r \u00a0phosphoproteomics\t\r \u00a0........................................................\t\r \u00a0112\t\r \u00a04.2.3\t\r \u00a0Mass\t\r \u00a0spectrometry\t\r \u00a0.........................................................................................................\t\r \u00a0112\t\r \u00a04.2.4\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0data\t\r \u00a0analysis\t\r \u00a0and\t\r \u00a0clustering\t\r \u00a0.......................................................................\t\r \u00a0112\t\r \u00a04.2.5\t\r \u00a0High-\u00ad\u2010throughput\t\r \u00a0western\t\r \u00a0blotting\t\r \u00a0..........................................................................\t\r \u00a0113\t\r \u00a04.3\t\r \u00a0Results\t\r \u00a0..........................................................................................................................................\t\r \u00a0114\t\r \u00a04.3.1\t\r \u00a0Quantitative\t\r \u00a0phosphoproteomic\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0signaling\t\r \u00a0during\t\r \u00a0 Salmonella\t\r \u00a0invasion\t\r \u00a0...................................................................................................................\t\r \u00a0114\t\r \u00a04.3.2\t\r \u00a0Validation\t\r \u00a0and\t\r \u00a0confirmation\t\r \u00a0of\t\r \u00a0phosphoproteomic\t\r \u00a0data\t\r \u00a0..............................\t\r \u00a0118\t\r \u00a04.3.3\t\r \u00a0Phosphorylation\t\r \u00a0dynamics\t\r \u00a0following\t\r \u00a0EGF\t\r \u00a0treatment\t\r \u00a0versus\t\r \u00a0Salmonella\t\r \u00a0infection\t\r \u00a0..........................................................................................................................................\t\r \u00a0120\t\r \u00a04.3.4\t\r \u00a0Salmonella\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0protein\t\r \u00a0SipB\t\r \u00a0is\t\r \u00a0phosphorylated\t\r \u00a0in\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0...................\t\r \u00a0123\t\r \u00a04.3.5\t\r \u00a0Novel\t\r \u00a0host\t\r \u00a0phosphorylation-\u00ad\u2010sites\t\r \u00a0regulated\t\r \u00a0during\t\r \u00a0Salmonella\t\r \u00a0infection\t\r \u00a0............................................................................................................................................................\t\r \u00a0124\t\r \u00a0 \t\r \u00a0 xi\t\r \u00a0 4.3.6\t\r \u00a0Phosphorylation\t\r \u00a0dynamics\t\r \u00a0following\t\r \u00a0WT\t\r \u00a0versus\t\r \u00a0\u2206sopB\t\r \u00a0infection\t\r \u00a0............\t\r \u00a0127\t\r \u00a04.4\t\r \u00a0Discussion\t\r \u00a0...................................................................................................................................\t\r \u00a0132\t\r \u00a05\t\r \u00a0Conclusion\t\r \u00a0...........................................................................................................................................\t\r \u00a0136\t\r \u00a0References\t\r \u00a0...............................................................................................................................................\t\r \u00a0143\t\r \u00a0Appendix\t\r \u00a0..................................................................................................................................................\t\r \u00a0184\t\r \u00a0Appendix\t\r \u00a0A\t\r \u00a0.........................................................................................................................................\t\r \u00a0184\t\r \u00a0 \t\r \u00a0 xii\t\r \u00a0 List\t\r \u00a0of\t\r \u00a0Tables\t\r \u00a0 Table\t\r \u00a01.1\t\r \u00a0Functions\t\r \u00a0and\t\r \u00a0host\t\r \u00a0targets\t\r \u00a0of\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0effectors\t\r \u00a0.....................................................\t\r \u00a016\t\r \u00a0Table\t\r \u00a01.2\t\r \u00a0Functions\t\r \u00a0and\t\r \u00a0host\t\r \u00a0targets\t\r \u00a0of\t\r \u00a0T3SS-\u00ad\u20102\t\r \u00a0effectors\t\r \u00a0.....................................................\t\r \u00a029\t\r \u00a0Table\t\r \u00a02.1\t\r \u00a0Summary\t\r \u00a0of\t\r \u00a0the\t\r \u00a0proteins\t\r \u00a0identified\t\r \u00a0by\t\r \u00a0MS\t\r \u00a0from\t\r \u00a0three\t\r \u00a0replicate\t\r \u00a0SILAC\t\r \u00a02HA-\u00ad\u2010SopB\t\r \u00a0immunoprecipitation\t\r \u00a0experiments\t\r \u00a0............................................................................\t\r \u00a071\t\r \u00a0Table\t\r \u00a04.1\t\r \u00a0Selected\t\r \u00a0novel\t\r \u00a0host\t\r \u00a0phosphosites\t\r \u00a0regulated\t\r \u00a0during\t\r \u00a0Salmonella\t\r \u00a0infection\t\r \u00a0............................................................................................................................................................\t\r \u00a0125\t\r \u00a0Table\t\r \u00a04.2\t\r \u00a0Kinases\t\r \u00a0most\t\r \u00a0frequently\t\r \u00a0predicted\t\r \u00a0upstream\t\r \u00a0of\t\r \u00a0upregulated\t\r \u00a0phosphosites\t\r \u00a0............................................................................................................................................................\t\r \u00a0126\t\r \u00a0\t\r \u00a0 \t\r \u00a0 xiii\t\r \u00a0 List\t\r \u00a0of\t\r \u00a0Figures\t\r \u00a0 Figure\t\r \u00a01.1\t\r \u00a0Diagram\t\r \u00a0of\t\r \u00a0SCV\t\r \u00a0biogenesis\t\r \u00a0and\t\r \u00a0maturation\t\r \u00a0.............................................................\t\r \u00a06\t\r \u00a0Figure\t\r \u00a01.2\t\r \u00a0Activity\t\r \u00a0flow\t\r \u00a0diagram\t\r \u00a0of\t\r 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\u00a0Figure\t\r \u00a02.4\t\r \u00a0Description\t\r \u00a0of\t\r \u00a02HA-\u00ad\u2010SopB\t\r \u00a0and\t\r \u00a0SopB-\u00ad\u2010GFP\t\r \u00a0and\t\r \u00a0GFP-\u00ad\u2010SopB\t\r \u00a0deletion\t\r \u00a0constructs\t\r \u00a0.........................................................................................................................................\t\r \u00a076\t\r \u00a0Figure\t\r \u00a02.5\t\r \u00a0Colocalization\t\r \u00a0of\t\r \u00a0SopB\t\r \u00a0with\t\r \u00a0Cdc42\t\r \u00a0............................................................................\t\r \u00a077\t\r \u00a0Figure\t\r \u00a02.6\t\r \u00a0Residues\t\r \u00a0117-\u00ad\u2010168\t\r \u00a0of\t\r \u00a0SopB\t\r \u00a0are\t\r \u00a0required\t\r \u00a0for\t\r \u00a0Cdc42\t\r \u00a0binding\t\r \u00a0...........................\t\r \u00a079\t\r \u00a0Figure\t\r \u00a02.7\t\r \u00a0The\t\r \u00a0SopB-\u00ad\u2010Cdc42\t\r \u00a0interaction\t\r \u00a0does\t\r \u00a0not\t\r \u00a0directly\t\r \u00a0activate\t\r \u00a0Cdc42\t\r \u00a0.....................\t\r \u00a080\t\r \u00a0Figure\t\r \u00a02.8\t\r \u00a0Ubiquitylation\t\r \u00a0peptides\t\r \u00a0identified\t\r \u00a0in\t\r \u00a02HA-\u00ad\u2010SopB\t\r \u00a0.................................................\t\r \u00a082\t\r \u00a0Figure\t\r \u00a03.1\t\r \u00a0Effects\t\r \u00a0of\t\r \u00a0phosphatase\t\r \u00a0inhibitors\t\r \u00a0during\t\r \u00a0cell\t\r \u00a0lysis\t\r \u00a0and\t\r \u00a0phosphopeptide\t\r \u00a0enrichment\t\r \u00a0.......................................................................................................................................\t\r \u00a098\t\r \u00a0Figure\t\r \u00a03.2\t\r \u00a0Phosphopeptide\t\r \u00a0enrichment\t\r \u00a0by\t\r \u00a0in-\u00ad\u2010solution\t\r \u00a0isoelectric\t\r \u00a0focusing\t\r \u00a0..............\t\r \u00a0100\t\r \u00a0Figure\t\r \u00a03.3\t\r \u00a0Phosphopeptide\t\r \u00a0enrichment\t\r \u00a0by\t\r \u00a0MOC\t\r \u00a0...................................................................\t\r \u00a0101\t\r \u00a0 \t\r \u00a0 xiv\t\r \u00a0 Figure\t\r \u00a03.4\t\r \u00a0Elution\t\r \u00a0times\t\r \u00a0of\t\r \u00a0phosphorylated\t\r \u00a0versus\t\r \u00a0non-\u00ad\u2010phosphorylated\t\r \u00a0peptides\t\r \u00a0from\t\r \u00a0a\t\r \u00a0C18\t\r \u00a0reversed\t\r \u00a0phase\t\r \u00a0column\t\r \u00a0......................................................................................\t\r \u00a0102\t\r \u00a0Figure\t\r \u00a03.5\t\r \u00a0Analyzing\t\r \u00a0phosphopeptides\t\r \u00a0by\t\r \u00a0LC-\u00ad\u2010ESI-\u00ad\u2010MSn\t\r \u00a0.......................................................\t\r \u00a0104\t\r \u00a0Figure\t\r \u00a04.1\t\r \u00a0Flow\t\r \u00a0diagram\t\r \u00a0outlining\t\r \u00a0phosphoproteomics\t\r \u00a0method\t\r \u00a0for\t\r \u00a0Salmonella\t\r \u00a0infection\t\r \u00a0..........................................................................................................................................\t\r \u00a0115\t\r \u00a0Figure\t\r \u00a04.2\t\r \u00a0Global\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0phosphoproteomics\t\r \u00a0data\t\r \u00a0.......................................................\t\r \u00a0117\t\r \u00a0Figure\t\r \u00a04.3\t\r \u00a0Validation\t\r \u00a0of\t\r \u00a0phosphoproteomics\t\r \u00a0data\t\r \u00a0................................................................\t\r \u00a0119\t\r \u00a0Figure\t\r \u00a04.4\t\r \u00a0Comparison\t\r \u00a0of\t\r \u00a0EGF-\u00ad\u2010\t\r \u00a0and\t\r \u00a0Salmonella-\u00ad\u2010induced\t\r \u00a0signaling\t\r \u00a0................................\t\r \u00a0122\t\r \u00a0Figure\t\r \u00a04.5\t\r \u00a0Novel\t\r \u00a0host\t\r \u00a0targets\t\r \u00a0of\t\r \u00a0the\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0effector\t\r \u00a0SopB\t\r \u00a0................................................\t\r \u00a0131\t\r \u00a0Figure\t\r \u00a04.6\t\r \u00a0Activity\t\r \u00a0flow\t\r \u00a0diagram\t\r \u00a0of\t\r \u00a0phosphorylation\t\r \u00a0based\t\r \u00a0signaling\t\r \u00a0cascades\t\r \u00a0initiated\t\r \u00a0by\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0effectors\t\r \u00a0.................................................................................................\t\r \u00a0135\t\r \u00a0\t\r \u00a0 \t\r \u00a0 xv\t\r \u00a0 List\t\r \u00a0of\t\r \u00a0Abbreviations\t\r \u00a0 2DE\t\r \u00a0 \t\r \u00a0 Two-\u00ad\u2010dimensional\t\r \u00a0gel\t\r \u00a0electrophoresis\t\r \u00a0AGC\t\r \u00a0 \t\r \u00a0 Automatic\t\r \u00a0gain\t\r \u00a0control\t\r \u00a0AHNAK\t\r \u00a0 Desmoyokin\t\r \u00a0Akt\t\r \u00a0 \t\r \u00a0 Protein\t\r \u00a0kinase\t\r \u00a0B\t\r \u00a0AP-\u00ad\u20101\t\r \u00a0 \t\r \u00a0 Activator\t\r \u00a0protein-\u00ad\u20101\t\r \u00a0ARAF\t\r \u00a0 \t\r \u00a0 Serine\/threonine-\u00ad\u2010protein\t\r \u00a0kinase\t\r \u00a0A-\u00ad\u2010Raf\t\r \u00a0ARF6\t\r \u00a0 \t\r \u00a0 ADP-\u00ad\u2010ribosylation\t\r \u00a0factor\t\r \u00a06\t\r \u00a0Arp2\/3\t\r \u00a0 Actin-\u00ad\u2010related\t\r \u00a0protein-\u00ad\u20102\/3\t\r \u00a0ATF\t\r \u00a0 \t\r \u00a0 Cyclic\t\r \u00a0AMP-\u00ad\u2010dependent\t\r \u00a0transcription\t\r \u00a0factor\t\r \u00a0BAD\t\r \u00a0 \t\r \u00a0 Bcl2\t\r \u00a0antagonist\t\r \u00a0of\t\r \u00a0cell\t\r \u00a0death\t\r \u00a0Cdc42\t\r \u00a0 \t\r \u00a0 Cell\t\r \u00a0division\t\r \u00a0control\t\r \u00a0protein\t\r \u00a042\t\r \u00a0CFL\t\r \u00a0 \t\r \u00a0 Cofilin\t\r \u00a0CID\t\r \u00a0 \t\r \u00a0 Collision-\u00ad\u2010induced\t\r \u00a0dissociation\t\r \u00a0CLK1\t\r \u00a0 \t\r \u00a0 Dual\t\r \u00a0specificity\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a0CLK1\t\r \u00a0CRK\t\r \u00a0 \t\r \u00a0 Cell\t\r \u00a0division\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a012\t\r \u00a0Da\t\r \u00a0 \t\r \u00a0 Dalton\t\r \u00a0DHB\t\r \u00a0 \t\r \u00a0 Dihydroxy-\u00ad\u2010benzoic\t\r \u00a0acid\t\r \u00a0DMEM\t\r \u00a0\t\r \u00a0 Dulbecco\u2019s\t\r \u00a0Modified\t\r \u00a0Eagle\t\r \u00a0Medium\t\r \u00a0ECD\t\r \u00a0 \t\r \u00a0 Electron\t\r \u00a0capture\t\r \u00a0dissociation\t\r \u00a0ECL\t\r \u00a0 \t\r \u00a0 Enhanced\t\r \u00a0chemiluminescence\t\r \u00a0EGFR\t\r \u00a0 \t\r \u00a0 Epidermal\t\r \u00a0growth\t\r \u00a0factor\t\r \u00a0receptor\t\r \u00a0EHBP1L1\t\r \u00a0 EH\t\r \u00a0domain-\u00ad\u2010binding\t\r \u00a0protein\t\r \u00a01-\u00ad\u2010like\t\r \u00a0protein\t\r \u00a01\t\r \u00a0 \t\r \u00a0 xvi\t\r \u00a0 ERdj3\t\r \u00a0 \t\r \u00a0 ER-\u00ad\u2010associated\t\r \u00a0dnaJ\t\r \u00a0protein\t\r \u00a03\t\r \u00a0ERK\t\r \u00a0 \t\r \u00a0 Extracellular\t\r \u00a0signal-\u00ad\u2010regulated\t\r \u00a0kinase\t\r \u00a0ERLIC\t\r \u00a0 \t\r \u00a0 Electrostatic\t\r \u00a0repulsion-\u00ad\u2010hydrophilic\t\r \u00a0interaction\t\r \u00a0chromatography\t\r \u00a0ESI\t\r \u00a0 \t\r \u00a0 Electrospray\t\r \u00a0ionization\t\r \u00a0ETD\t\r \u00a0 \t\r \u00a0 Electron\t\r \u00a0transfer\t\r \u00a0dissociation\t\r \u00a0FBS\t\r \u00a0 \t\r \u00a0 Fetal\t\r \u00a0bovine\t\r \u00a0serum\t\r \u00a0GAP\t\r \u00a0 \t\r \u00a0 GTPase-\u00ad\u2010activating\t\r \u00a0protein\t\r \u00a0GEF\t\r \u00a0 \t\r \u00a0 Guanine\t\r \u00a0nucleotide\t\r \u00a0exchange\t\r \u00a0factor\t\r \u00a0GFP\t\r \u00a0 \t\r \u00a0 Green\t\r \u00a0fluorescent\t\r \u00a0protein\t\r \u00a0GO\t\r \u00a0 \t\r \u00a0 Gene\t\r \u00a0Ontology\t\r \u00a0GPI-\u00ad\u2010AP\t\r \u00a0 Glycosylphosphatidylinositol-\u00ad\u2010anchored\t\r \u00a0proteins\t\r \u00a0GST\t\r \u00a0 \t\r \u00a0 Glutathione\t\r \u00a0S-\u00ad\u2010transferase\t\r \u00a0GTPase\t\r \u00a0 Guanosine\t\r \u00a0triphosphatase\t\r \u00a0H\t\r \u00a0 \t\r \u00a0 Hour\t\r \u00a0HA\t\r \u00a0 \t\r \u00a0 Hemagglutinin\t\r \u00a0HCD\t\r \u00a0 \t\r \u00a0 Higher-\u00ad\u2010energy\t\r \u00a0c-\u00ad\u2010trap\t\r \u00a0dissociation\t\r \u00a0HDAC1\t\r \u00a0 Histone\t\r \u00a0deacetylase\t\r \u00a01\t\r \u00a0HILIC\t\r \u00a0 \t\r \u00a0 Hydrophilic\t\r \u00a0interaction\t\r \u00a0chromatography\t\r \u00a0HMG14\t\r \u00a0 Non-\u00ad\u2010histone\t\r \u00a0chromosomal\t\r \u00a0protein\t\r \u00a0HMG-\u00ad\u201014\t\r \u00a0Hook3\t\r \u00a0\t\r \u00a0 Protein\t\r \u00a0Hook\t\r \u00a0homolog\t\r \u00a03\t\r \u00a0HPLC\t\r \u00a0 \t\r \u00a0 High-\u00ad\u2010performance\t\r \u00a0liquid\t\r \u00a0chromatography\t\r \u00a0ICAT\t\r \u00a0 \t\r \u00a0 Isotope-\u00ad\u2010coded\t\r \u00a0affinity\t\r \u00a0tagging\t\r \u00a0IEF\t\r \u00a0 \t\r \u00a0 Isoeletric\t\r \u00a0focusing\t\r \u00a0 \t\r \u00a0 xvii\t\r \u00a0 I-\u00ad\u2010\u03baB\u03b1\t\r \u00a0 \t\r \u00a0 NF-\u00ad\u2010\u03baB\t\r \u00a0inhibitor\t\r \u00a0alpha\t\r \u00a0IKK\t\r \u00a0 \t\r \u00a0 I-\u00ad\u2010\u03baB\t\r \u00a0kinase\t\r \u00a0IL-\u00ad\u20108\t\r \u00a0 \t\r \u00a0 Interleukin\t\r \u00a08\t\r \u00a0IMAC\t\r \u00a0 \t\r \u00a0 Immobilized\t\r \u00a0metal\t\r \u00a0affinity\t\r \u00a0chromatography\t\r \u00a0IPG\t\r \u00a0 \t\r \u00a0 Immobilized\t\r \u00a0pH\t\r \u00a0gradient\t\r \u00a0IQGAP1\t\r \u00a0 Ras\t\r \u00a0GTPase-\u00ad\u2010activating-\u00ad\u2010like\t\r \u00a0protein\t\r \u00a0IQGAP1\t\r \u00a0ITRAQ\t\r \u00a0\t\r \u00a0 Isotope\t\r \u00a0tags\t\r \u00a0for\t\r \u00a0relative\t\r \u00a0and\t\r \u00a0absolute\t\r \u00a0quantification\t\r \u00a0JNK\t\r \u00a0 \t\r \u00a0 C-\u00ad\u2010Jun\t\r \u00a0N-\u00ad\u2010terminal\t\r \u00a0kinase\t\r \u00a0KANK2\t\r \u00a0 KN\t\r \u00a0motif\t\r \u00a0and\t\r \u00a0ankyrin\t\r \u00a0repeat\t\r \u00a0domain-\u00ad\u2010containing\t\r \u00a0protein\t\r \u00a02\t\r \u00a0KB\t\r \u00a0 \t\r \u00a0 Kilo\t\r \u00a0base\t\r \u00a0pairs\t\r \u00a0KIF4A\t\r \u00a0 \t\r \u00a0 Chromosome-\u00ad\u2010associated\t\r \u00a0kinesin\t\r \u00a0KIF4A\t\r \u00a0LAMP\t\r \u00a0 \t\r \u00a0 Lysosomal-\u00ad\u2010associated\t\r \u00a0membrane\t\r \u00a0protein\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0 Liquid\t\r \u00a0chromatography-\u00ad\u2010tandem\t\r \u00a0mass\t\r \u00a0spectrometry\t\r \u00a0LIMK\t\r \u00a0 \t\r \u00a0 LIM\t\r \u00a0domain\t\r \u00a0kinase\t\r \u00a01\t\r \u00a0LMO7\t\r \u00a0 \t\r \u00a0 LIM\t\r \u00a0domain\t\r \u00a0only\t\r \u00a0protein\t\r \u00a07\t\r \u00a0M\t\r \u00a0cell\t\r \u00a0 \t\r \u00a0 Microfold\t\r \u00a0cell\t\r \u00a0M\/Z\t\r \u00a0 \t\r \u00a0 Mass\t\r \u00a0to\t\r \u00a0charge\t\r \u00a0ratio\t\r \u00a0M6PR\t\r \u00a0 \t\r \u00a0 Mannose-\u00ad\u20106-\u00ad\u2010phosphate\t\r \u00a0receptor\t\r \u00a0MAPK\t\r \u00a0 \t\r \u00a0 Mitogen-\u00ad\u2010activated\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a0MEK\t\r \u00a0 \t\r \u00a0 Dual\t\r \u00a0specificity\t\r \u00a0mitogen-\u00ad\u2010activated\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a0kinase\t\r \u00a0MEKK\t\r \u00a0 \t\r \u00a0 Dual\t\r \u00a0specificity\t\r \u00a0mitogen-\u00ad\u2010activated\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a0kinase\t\r \u00a0kinase\t\r \u00a0Min\t\r \u00a0 \t\r \u00a0 Minute\t\r \u00a0MOC\t\r \u00a0 \t\r \u00a0 Metal\t\r \u00a0oxide\t\r \u00a0chromatography\t\r \u00a0 \t\r \u00a0 xviii\t\r \u00a0 MOI\t\r \u00a0 \t\r \u00a0 Multiplicity\t\r \u00a0of\t\r \u00a0infection\t\r \u00a0MSK2\t\r \u00a0 \t\r \u00a0 Ribosomal\t\r \u00a0protein\t\r \u00a0S6\t\r \u00a0kinase\t\r \u00a0alpha-\u00ad\u20104\t\r \u00a0MTOR\t\r \u00a0 \t\r \u00a0 Mammalian\t\r \u00a0target\t\r \u00a0of\t\r \u00a0rapamycin\t\r \u00a0NHE1\t\r \u00a0 \t\r \u00a0 Sodium\/hydrogen\t\r \u00a0exchanger\t\r \u00a01\t\r \u00a0NF-\u00ad\u2010\u03baB\t\r \u00a0\t\r \u00a0 Nuclear\t\r \u00a0factor-\u00ad\u2010kB\t\r \u00a0NLR\t\r \u00a0 \t\r \u00a0 Nucleotide\t\r \u00a0oligomerization\t\r \u00a0domain-\u00ad\u2010like\t\r \u00a0receptor\t\r \u00a0P38\t\r \u00a0 \t\r \u00a0 Mitogen-\u00ad\u2010activated\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a0p38\t\r \u00a0P70S6K\t\r \u00a0\t\r \u00a0 Ribosomal\t\r \u00a0protein\t\r \u00a0S6\t\r \u00a0kinase\t\r \u00a0beta\t\r \u00a0PAK4\t\r \u00a0 \t\r \u00a0 P21-\u00ad\u2010activated\t\r \u00a0kinase\t\r \u00a04\t\r \u00a0PAMP\t\r \u00a0 \t\r \u00a0 Pathogen-\u00ad\u2010associated\t\r \u00a0molecular\t\r \u00a0pattern\t\r \u00a0PBS\t\r \u00a0 \t\r \u00a0 Phosphate-\u00ad\u2010buffered\t\r \u00a0saline\t\r \u00a0PFKFB2\t\r \u00a0 6-\u00ad\u2010phosphofructo-\u00ad\u20102-\u00ad\u2010kinase\/fructose-\u00ad\u20102,6-\u00ad\u2010biphosphatase\t\r \u00a02\t\r \u00a0PHF14\t\r \u00a0\t\r \u00a0 PHD\t\r \u00a0finger\t\r \u00a0protein\t\r \u00a014\t\r \u00a0PI\t\r \u00a0 \t\r \u00a0 Isoelectric\t\r \u00a0point\t\r \u00a0PI3P\t\r \u00a0 \t\r \u00a0 Phosphatidylinositol\t\r \u00a03-\u00ad\u2010phosphate\t\r \u00a0PIKFYVE\t\r \u00a0 1-\u00ad\u2010phosphatidylinositol-\u00ad\u20103-\u00ad\u2010phosphate\t\r \u00a05-\u00ad\u2010kinase\t\r \u00a0PIM\t\r \u00a0 \t\r \u00a0 Proto-\u00ad\u2010oncogene\t\r \u00a0serine\/threonine-\u00ad\u2010protein\t\r \u00a0kinase\t\r \u00a0pim\t\r \u00a0PI(4,5)P2\t\r \u00a0 Phosphatidylinositol\t\r \u00a04,5-\u00ad\u2010bisphosphate\"\t\r \u00a0PKC\t\r \u00a0 \t\r \u00a0 Protein\t\r \u00a0kinase\t\r \u00a0C\t\r \u00a0PKN1\t\r \u00a0 \t\r \u00a0 Serine\/threonine\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a0N1\t\r \u00a0PLEC1\t\r \u00a0\t\r \u00a0 Plectin\t\r \u00a0PMN\t\r \u00a0 \t\r \u00a0 Polymorphonuclear\t\r \u00a0leukocyte\t\r \u00a0PP\t\r \u00a0 \t\r \u00a0 Protein\t\r \u00a0phosphatase\t\r \u00a0 \t\r \u00a0 xix\t\r \u00a0 PRKD3\t\r \u00a0 Protein-\u00ad\u2010serine\/threonine\t\r \u00a0kinase\t\r \u00a0D3\t\r \u00a0PS\t\r \u00a0 \t\r \u00a0 Phosphoserine\t\r \u00a0PSKH1\t\r \u00a0\t\r \u00a0 Serine\/threonine-\u00ad\u2010protein\t\r \u00a0kinase\t\r \u00a0H1\t\r \u00a0PT\t\r \u00a0 \t\r \u00a0 Phosphothreonine\t\r \u00a0PTM\t\r \u00a0 \t\r \u00a0 Post-\u00ad\u2010translational\t\r \u00a0modification\t\r \u00a0PTP\t\r \u00a0 \t\r \u00a0 Protein\t\r \u00a0tyrosine\t\r \u00a0phosphatase\t\r \u00a0PY\t\r \u00a0 \t\r \u00a0 Phosphotyrosine\t\r \u00a0Rab\t\r \u00a0 \t\r \u00a0 Ras-\u00ad\u2010related\t\r \u00a0protein\t\r \u00a0Rac1\t\r \u00a0 \t\r \u00a0 Ras-\u00ad\u2010related\t\r \u00a0C3\t\r \u00a0bolulinum\t\r \u00a0toxin\t\r \u00a0substrate\t\r \u00a01\t\r \u00a0Raf1\t\r \u00a0 \t\r \u00a0 RAF\t\r \u00a0proto-\u00ad\u2010oncogene\t\r \u00a0serine\/threonine-\u00ad\u2010protein\t\r \u00a0kinase\t\r \u00a0RCF\t\r \u00a0 \t\r \u00a0 Relative\t\r \u00a0centrifugal\t\r \u00a0force\t\r \u00a0Rho\t\r \u00a0 \t\r \u00a0 Rho-\u00ad\u2010related\t\r \u00a0GTP-\u00ad\u2010binding\t\r \u00a0protein\t\r \u00a0RILP\t\r \u00a0 \t\r \u00a0 Rab7-\u00ad\u2010interacting\t\r \u00a0lysosomal\t\r \u00a0protein\t\r \u00a0ROCK2\t\r \u00a0 Rho-\u00ad\u2010associated\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a02\t\r \u00a0RPM\t\r \u00a0 \t\r \u00a0 Revolutions\t\r \u00a0per\t\r \u00a0minute\t\r \u00a0RPS6\t\r \u00a0 \t\r \u00a0 40S\t\r \u00a0ribosomal\t\r \u00a0protein\t\r \u00a0S6\t\r \u00a0RSK1\t\r \u00a0 \t\r \u00a0 Ribosomal\t\r \u00a0protein\t\r \u00a0S6\t\r \u00a0kinase\t\r \u00a0alpha-\u00ad\u20101\t\r \u00a0RT\t\r \u00a0 \t\r \u00a0 Room\t\r \u00a0temperature\t\r \u00a0SAX\t\r \u00a0 \t\r \u00a0 Strong\t\r \u00a0anion\t\r \u00a0exchange\t\r \u00a0SCAMP3\t\r \u00a0 Secretary\t\r \u00a0carrier-\u00ad\u2010associated\t\r \u00a0membrane\t\r \u00a0protein\t\r \u00a03\t\r \u00a0SCV\t\r \u00a0 \t\r \u00a0 Salmonella-\u00ad\u2010containing\t\r \u00a0vacuole\t\r \u00a0SCX\t\r \u00a0 \t\r \u00a0 Strong\t\r \u00a0cation\t\r \u00a0exchange\t\r \u00a0SDS-\u00ad\u2010PAGE\t\r \u00a0 Sodium\t\r \u00a0dodecyl\t\r \u00a0sulfate-\u00ad\u2010polyacrylamide\t\r \u00a0gel\t\r \u00a0electrophoresis\t\r \u00a0 \t\r \u00a0 xx\t\r \u00a0 Sec\t\r \u00a0 \t\r \u00a0 Seconds\t\r \u00a0SEX\t\r \u00a0 \t\r \u00a0 Size\t\r \u00a0exclusion\t\r \u00a0SGEF\t\r \u00a0 \t\r \u00a0 RhoG\t\r \u00a0through\t\r \u00a0SH3-\u00ad\u2010containing\t\r \u00a0GEF\t\r \u00a0SGK\t\r \u00a0 \t\r \u00a0 Serum\/glucocorticoid-\u00ad\u2010regulated\t\r \u00a0kinase\t\r \u00a0SIF\t\r \u00a0 \t\r \u00a0 Salmonella-\u00ad\u2010induced\t\r \u00a0filament\t\r \u00a0SILAC\t\r \u00a0 \t\r \u00a0 Stable\t\r \u00a0isotope\t\r \u00a0labeling\t\r \u00a0by\t\r \u00a0amino\t\r \u00a0acids\t\r \u00a0in\t\r \u00a0cell\t\r \u00a0culture\t\r \u00a0SIMAC\t\r \u00a0\t\r \u00a0 Sequential\t\r \u00a0Elution\t\r \u00a0from\t\r \u00a0IMAC\t\r \u00a0SKIP\t\r \u00a0 \t\r \u00a0 SifA\t\r \u00a0and\t\r \u00a0kinesin-\u00ad\u2010interacting\t\r \u00a0protein\t\r \u00a0SNX-\u00ad\u20101\t\r \u00a0 \t\r \u00a0 Sorting\t\r \u00a0nexin-\u00ad\u20101\t\r \u00a0SNX-\u00ad\u20103\t\r \u00a0 \t\r \u00a0 Sorting\t\r \u00a0nexin-\u00ad\u20103\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0 \t\r \u00a0 Salmonella\t\r \u00a0pathogenicity\t\r \u00a0island\t\r \u00a01\t\r \u00a0SPI-\u00ad\u20102\t\r \u00a0 \t\r \u00a0 Salmonella\t\r \u00a0pathogenicity\t\r \u00a0island\t\r \u00a02\t\r \u00a0Src\t\r \u00a0 \t\r \u00a0 Proto-\u00ad\u2010oncogene\t\r \u00a0tyrosine-\u00ad\u2010protein\t\r \u00a0kinase\t\r \u00a0Src\t\r \u00a0STAGE\t\r \u00a0\t\r \u00a0 STop\t\r \u00a0And\t\r \u00a0Go\t\r \u00a0Extraction\t\r \u00a0STX7\t\r \u00a0 \t\r \u00a0 Syntaxin-\u00ad\u20107\t\r \u00a0T3SS\t\r \u00a0 \t\r \u00a0 Type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0system\t\r \u00a0TassC\t\r \u00a0 \t\r \u00a0 Target\t\r \u00a0for\t\r \u00a0Salmonella\t\r \u00a0secreted\t\r \u00a0protein\t\r \u00a0C\t\r \u00a0TFA\t\r \u00a0 \t\r \u00a0 Trifluoroacetic\t\r \u00a0acid\t\r \u00a0TGN\t\r \u00a0 \t\r \u00a0 Trans-\u00ad\u2010Golgi\t\r \u00a0network\t\r \u00a0Th\t\r \u00a0 \t\r \u00a0 Thomson\t\r \u00a0TLR\t\r \u00a0 \t\r \u00a0 Toll-\u00ad\u2010like\t\r \u00a0receptor\t\r \u00a0TRIP6\t\r \u00a0 \t\r \u00a0 Thyroid\t\r \u00a0receptor-\u00ad\u2010interacting\t\r \u00a0protein\t\r \u00a06\t\r \u00a0V-\u00ad\u2010ATPase\t\r \u00a0 Vacuolar-\u00ad\u2010type\t\r \u00a0H+-\u00ad\u2010ATPase\t\r \u00a0 \t\r \u00a0 xxi\t\r \u00a0 VAMP8\t\r \u00a0 Vesicle-\u00ad\u2010associated\t\r \u00a0membrane\t\r \u00a0protein\t\r \u00a08\t\r \u00a0VAP\t\r \u00a0 \t\r \u00a0 Vacuole-\u00ad\u2010associated\t\r \u00a0actin\t\r \u00a0polymerization\t\r \u00a0VCP\t\r \u00a0 \t\r \u00a0 Transitional\t\r \u00a0endoplasmic\t\r \u00a0reticulum\t\r \u00a0ATPase\t\r \u00a0Vps34\t\r \u00a0\t\r \u00a0 Phosphatidylinositol\t\r \u00a03-\u00ad\u2010kinase\t\r \u00a0catalytic\t\r \u00a0subunit\t\r \u00a0type\t\r \u00a03\t\r \u00a0WASP\t\r \u00a0 \t\r \u00a0 Wiskott-\u00ad\u2010Aldrich\t\r \u00a0syndrome\t\r \u00a0protein\t\r \u00a0WT\t\r \u00a0 \t\r \u00a0 Wild\t\r \u00a0type\t\r \u00a0 XCV\t\r \u00a0 \t\r \u00a0 Average\t\r \u00a0coefficient\t\r \u00a0of\t\r \u00a0variation\t\r \u00a0 \t\r \u00a0 xxii\t\r \u00a0 Acknowledgements\t\r \u00a0 I\t\r \u00a0would\t\r \u00a0like\t\r \u00a0to\t\r \u00a0thank\t\r \u00a0my\t\r \u00a0supervisor\t\r \u00a0Dr.\t\r \u00a0L.J.\t\r \u00a0Foster\t\r \u00a0for\t\r \u00a0teaching\t\r \u00a0me\t\r \u00a0to\t\r \u00a0become\t\r \u00a0a\t\r \u00a0scientist\t\r \u00a0and\t\r \u00a0for\t\r \u00a0supporting\t\r \u00a0me\t\r \u00a0throughout\t\r \u00a0my\t\r \u00a0graduate\t\r \u00a0studies\t\r \u00a0at\t\r \u00a0UBC.\t\r \u00a0Thank-\u00ad\u2010you\t\r \u00a0for\t\r \u00a0allowing\t\r \u00a0me\t\r \u00a0to\t\r \u00a0create\t\r \u00a0and\t\r \u00a0follow\t\r \u00a0on\t\r \u00a0my\t\r \u00a0own\t\r \u00a0ideas\t\r \u00a0and\t\r \u00a0interests,\t\r \u00a0and\t\r \u00a0for\t\r \u00a0your\t\r \u00a0endless\t\r \u00a0support\t\r \u00a0of\t\r \u00a0both\t\r \u00a0my\t\r \u00a0development\t\r \u00a0as\t\r \u00a0a\t\r \u00a0scientist,\t\r \u00a0and\t\r \u00a0my\t\r \u00a0life\t\r \u00a0outside\t\r \u00a0the\t\r \u00a0lab.\t\r \u00a0Thanks\t\r \u00a0also\t\r \u00a0to\t\r \u00a0my\t\r \u00a0fellow\t\r \u00a0lab\t\r \u00a0members\t\r \u00a0for\t\r \u00a0your\t\r \u00a0guidance\t\r \u00a0and\t\r \u00a0support,\t\r \u00a0and\t\r \u00a0for\t\r \u00a0all\t\r \u00a0the\t\r \u00a0fun\t\r \u00a0we\u2019ve\t\r \u00a0shared\t\r \u00a0during\t\r \u00a0the\t\r \u00a0past\t\r \u00a0five\t\r \u00a0years.\t\r \u00a0I\t\r \u00a0especially\t\r \u00a0thank\t\r \u00a0Queenie\t\r \u00a0Chan\t\r \u00a0for\t\r \u00a0her\t\r \u00a0advice\t\r \u00a0and\t\r \u00a0support\t\r \u00a0throughout,\t\r \u00a0and\t\r \u00a0Nikolay\t\r \u00a0Stoynov\t\r \u00a0for\t\r \u00a0his\t\r \u00a0patience\t\r \u00a0and\t\r \u00a0technical\t\r \u00a0assistance.\t\r \u00a0Thank-\u00ad\u2010you\t\r \u00a0to\t\r \u00a0my\t\r \u00a0supervisory\t\r \u00a0committee\t\r \u00a0members:\t\r \u00a0Dr.\t\r \u00a0B.E.\t\r \u00a0Ellis,\t\r \u00a0Dr.\t\r \u00a0B.B.\t\r \u00a0Finlay\t\r \u00a0and\t\r \u00a0Dr.\t\r \u00a0C.M.\t\r \u00a0Overall.\t\r \u00a0I\t\r \u00a0would\t\r \u00a0especially\t\r \u00a0like\t\r \u00a0to\t\r \u00a0Dr.\t\r \u00a0B.B.\t\r \u00a0Finlay\t\r \u00a0for\t\r \u00a0his\t\r \u00a0advice\t\r \u00a0and\t\r \u00a0support\t\r \u00a0pertaining\t\r \u00a0to\t\r \u00a0Salmonella\t\r \u00a0biology,\t\r \u00a0and\t\r \u00a0Dr.\t\r \u00a0B.E.\t\r \u00a0Ellis\t\r \u00a0and\t\r \u00a0Dr.\t\r \u00a0C.M.\t\r \u00a0Overall\t\r \u00a0for\t\r \u00a0advice\t\r \u00a0on\t\r \u00a0cell\t\r \u00a0signaling\t\r \u00a0and\t\r \u00a0proteomics\t\r \u00a0techniques,\t\r \u00a0and\t\r \u00a0for\t\r \u00a0supporting\t\r \u00a0my\t\r \u00a0fellowship\t\r \u00a0applications.\t\r \u00a0Finally,\t\r \u00a0I\t\r \u00a0offer\t\r \u00a0many\t\r \u00a0thanks\t\r \u00a0to\t\r \u00a0my\t\r \u00a0family.\t\r \u00a0Thanks\t\r \u00a0to\t\r \u00a0my\t\r \u00a0Mom\t\r \u00a0and\t\r \u00a0Dad\t\r \u00a0for\t\r \u00a0believing\t\r \u00a0in\t\r \u00a0me\t\r \u00a0and\t\r \u00a0for\t\r \u00a0giving\t\r \u00a0me\t\r \u00a0the\t\r \u00a0confidence\t\r \u00a0to\t\r \u00a0leave\t\r \u00a0our\t\r \u00a0small\t\r \u00a0town\t\r \u00a0and\t\r \u00a0go\t\r \u00a0and\t\r \u00a0do\t\r \u00a0and\t\r \u00a0study\t\r \u00a0whatever\t\r \u00a0I\t\r \u00a0wished.\t\r \u00a0Thanks\t\r \u00a0also\t\r \u00a0to\t\r \u00a0my\t\r \u00a0brother\t\r \u00a0Kevin\t\r \u00a0for\t\r \u00a0always\t\r \u00a0being\t\r \u00a0so\t\r \u00a0proud\t\r \u00a0of\t\r \u00a0me.\t\r \u00a0I\t\r \u00a0would\t\r \u00a0lastly\t\r \u00a0like\t\r \u00a0to\t\r \u00a0thank\t\r \u00a0my\t\r \u00a0husband\t\r \u00a0Nat.\t\r \u00a0You\t\r \u00a0are\t\r \u00a0everything\t\r \u00a0to\t\r \u00a0me.\t\r \u00a0You\t\r \u00a0are\t\r \u00a0my\t\r \u00a0best\t\r \u00a0friend,\t\r \u00a0my\t\r \u00a0advisor,\t\r \u00a0and\t\r \u00a0my\t\r \u00a0confidant,\t\r \u00a0and\t\r \u00a0you\t\r \u00a0have\t\r \u00a0helped\t\r \u00a0me\t\r \u00a0through\t\r \u00a0everything\t\r \u00a0I\t\r \u00a0have\t\r \u00a0done\t\r \u00a0here.\t\r \u00a0 \t\r \u00a0 xxiii\t\r \u00a0 Dedication\t\r \u00a0 To my parents, my brother Kevin and Nat. \t\r \u00a0 \t\r \u00a0\t\r \u00a0 1\t\r \u00a0 1\t\r \u00a0Introduction\t\r \u00a0 1.1\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0 1.1.1\t\r \u00a0Salmonella\t\r \u00a0phylogeny\t\r \u00a0 Salmonella\t\r \u00a0is\t\r \u00a0a\t\r \u00a0genus\t\r \u00a0of\t\r \u00a0rod-\u00ad\u2010shaped,\t\r \u00a0Gram\t\r \u00a0negative,\t\r \u00a0facultative\t\r \u00a0anaerobic\t\r \u00a0bacteria.\t\r \u00a0Two\t\r \u00a0species\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0exist,\t\r \u00a0termed\t\r \u00a0Salmonella\t\r \u00a0bongori\t\r \u00a0and\t\r \u00a0Salmonella\t\r \u00a0 enterica.\t\r \u00a0While\t\r \u00a0S.\t\r \u00a0bongori\t\r \u00a0is\t\r \u00a0non-\u00ad\u2010pathogenic\t\r \u00a0for\t\r \u00a0mammals,\t\r \u00a0thousands\t\r \u00a0of\t\r \u00a0serovars\t\r \u00a0of\t\r \u00a0 S.\t\r \u00a0enterica\t\r \u00a0exist\t\r \u00a0which\t\r \u00a0can\t\r \u00a0cause\t\r \u00a0various\t\r \u00a0manifestations\t\r \u00a0of\t\r \u00a0disease(1).\t\r \u00a0All\t\r \u00a0Salmonella\t\r \u00a0 enterica\t\r \u00a0serovars\t\r \u00a0are\t\r \u00a0pathogenic\t\r \u00a0to\t\r \u00a0humans,\t\r \u00a0and\t\r \u00a0they\t\r \u00a0are\t\r \u00a0classified\t\r \u00a0into\t\r \u00a0subspecies\t\r \u00a0according\t\r \u00a0to\t\r \u00a0the\t\r \u00a0Kaufman-\u00ad\u2010White\t\r \u00a0serotyping\t\r \u00a0scheme,\t\r \u00a0which\t\r \u00a0is\t\r \u00a0based\t\r \u00a0on\t\r \u00a0lipopolysaccharide,\t\r \u00a0flagellar\t\r \u00a0and\t\r \u00a0capsular\t\r \u00a0antigens.\t\r \u00a0\t\r \u00a0However,\t\r \u00a0they\t\r \u00a0are\t\r \u00a0often\t\r \u00a0additionally\t\r \u00a0classified\t\r \u00a0according\t\r \u00a0to\t\r \u00a0their\t\r \u00a0host\t\r \u00a0adaptation\t\r \u00a0and\t\r \u00a0a\t\r \u00a0few\t\r \u00a0have\t\r \u00a0a\t\r \u00a0limited\t\r \u00a0host\t\r \u00a0spectrum\t\r \u00a0in\t\r \u00a0other\t\r \u00a0mammals.\t\r \u00a0For\t\r \u00a0example,\t\r \u00a0Salmonella\t\r \u00a0Typhi\t\r \u00a0and\t\r \u00a0Paratyphi\t\r \u00a0infect\t\r \u00a0only\t\r \u00a0primates,\t\r \u00a0Salmonella\t\r \u00a0Dublin\t\r \u00a0infects\t\r \u00a0only\t\r \u00a0cattle,\t\r \u00a0and\t\r \u00a0Salmonella\t\r \u00a0Choleraesuis\t\r \u00a0infects\t\r \u00a0only\t\r \u00a0pigs.\t\r \u00a0In\t\r \u00a0humans,\t\r \u00a0the\t\r \u00a0most\t\r \u00a0common\t\r \u00a0serovars\t\r \u00a0are\t\r \u00a0Typhimurium\t\r \u00a0and\t\r \u00a0Enteriditis,\t\r \u00a0both\t\r \u00a0of\t\r \u00a0which\t\r \u00a0case\t\r \u00a0gastroenteritis.\t\r \u00a0S.\t\r \u00a0Typhi\t\r \u00a0and\t\r \u00a0S.\t\r \u00a0Paratyphi\t\r \u00a0infections\t\r \u00a0are\t\r \u00a0less\t\r \u00a0common,\t\r \u00a0but\t\r \u00a0cause\t\r \u00a0a\t\r \u00a0more\t\r \u00a0severe\t\r \u00a0infection\t\r \u00a0termed\t\r \u00a0enteric\t\r \u00a0or\t\r \u00a0typhoid\t\r \u00a0fever.\t\r \u00a0In\t\r \u00a0addition,\t\r \u00a0S.\t\r \u00a0Typhimurium\t\r \u00a0causes\t\r \u00a0enteric\t\r \u00a0fever\t\r \u00a0in\t\r \u00a0mice\t\r \u00a0and\t\r \u00a0is\t\r \u00a0commonly\t\r \u00a0used\t\r \u00a0in\t\r \u00a0research\t\r \u00a0as\t\r \u00a0a\t\r \u00a0model\t\r \u00a0for\t\r \u00a0human\t\r \u00a0typhoid\t\r \u00a0fever.\t\r \u00a0\t\r \u00a0 1.1.2\t\r \u00a0Salmonella\t\r \u00a0and\t\r \u00a0human\t\r \u00a0health\t\r \u00a0 Salmonella\t\r \u00a0infections\t\r \u00a0pose\t\r \u00a0a\t\r \u00a0significant\t\r \u00a0health\t\r \u00a0burden\t\r \u00a0worldwide.\t\r \u00a0Non-\u00ad\u2010typhoid\t\r \u00a0Salmonella\t\r \u00a0infections\t\r \u00a0are\t\r \u00a0the\t\r \u00a0leading\t\r \u00a0cause\t\r \u00a0of\t\r \u00a0food\t\r \u00a0borne\t\r \u00a0illness\t\r \u00a0in\t\r \u00a0the\t\r \u00a0United\t\r \u00a0States\t\r \u00a0of\t\r \u00a0America,\t\r \u00a0and\t\r \u00a0globally\t\r \u00a0result\t\r \u00a0in\t\r \u00a01.3\t\r \u00a0billion\t\r \u00a0infections\t\r \u00a0annually(2).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 2\t\r \u00a0 Alternatively,\t\r \u00a0while\t\r \u00a0S.\t\r \u00a0Typhi\t\r \u00a0and\t\r \u00a0S.\t\r \u00a0Paratyphi\t\r \u00a0infections\t\r \u00a0have\t\r \u00a0been\t\r \u00a0practically\t\r \u00a0eliminated\t\r \u00a0in\t\r \u00a0the\t\r \u00a0industrial\t\r \u00a0world,\t\r \u00a0they\t\r \u00a0continue\t\r \u00a0to\t\r \u00a0pose\t\r \u00a0a\t\r \u00a0significant\t\r \u00a0health\t\r \u00a0problem\t\r \u00a0in\t\r \u00a0developing\t\r \u00a0countries.\t\r \u00a0In\t\r \u00a0these\t\r \u00a0regions,\t\r \u00a0approximately\t\r \u00a022\t\r \u00a0million\t\r \u00a0cases\t\r \u00a0of\t\r \u00a0typhoid\t\r \u00a0fever\t\r \u00a0are\t\r \u00a0reported\t\r \u00a0each\t\r \u00a0year,\t\r \u00a0resulting\t\r \u00a0in\t\r \u00a0216,000\t\r \u00a0deaths(3,\t\r \u00a04).\t\r \u00a0Furthermore,\t\r \u00a0while\t\r \u00a0fatality\t\r \u00a0rates\t\r \u00a0due\t\r \u00a0to\t\r \u00a0typhoid\t\r \u00a0fever\t\r \u00a0decreased\t\r \u00a0from\t\r \u00a010-\u00ad\u201030%\t\r \u00a0to\t\r \u00a01-\u00ad\u20104%\t\r \u00a0following\t\r \u00a0the\t\r \u00a0development\t\r \u00a0of\t\r \u00a0antimicrobials,\t\r \u00a0widespread\t\r \u00a0resistance\t\r \u00a0is\t\r \u00a0now\t\r \u00a0common\t\r \u00a0among\t\r \u00a0Salmonella\t\r \u00a0isolates(3).\t\r \u00a0Initially,\t\r \u00a0first\t\r \u00a0line\t\r \u00a0antibiotics\t\r \u00a0such\t\r \u00a0as\t\r \u00a0ampicillin,\t\r \u00a0trimethoprim-\u00ad\u2010sulphamethoxazole\t\r \u00a0and\t\r \u00a0chloramphenicol\t\r \u00a0were\t\r \u00a0effectively\t\r \u00a0used\t\r \u00a0to\t\r \u00a0treat\t\r \u00a0Salmonella\t\r \u00a0infections.\t\r \u00a0However,\t\r \u00a0following\t\r \u00a0heavy\t\r \u00a0use\t\r \u00a0for\t\r \u00a0human\t\r \u00a0treatment\t\r \u00a0and\t\r \u00a0especially\t\r \u00a0heavy\t\r \u00a0use\t\r \u00a0in\t\r \u00a0animals\t\r \u00a0for\t\r \u00a0food\t\r \u00a0production,\t\r \u00a0resistance\t\r \u00a0to\t\r \u00a0these\t\r \u00a0antimicrobials\t\r \u00a0is\t\r \u00a0now\t\r \u00a0widespread.\t\r \u00a0Multidrug\t\r \u00a0resistance\t\r \u00a0(defined\t\r \u00a0as\t\r \u00a0resistance\t\r \u00a0to\t\r \u00a0these\t\r \u00a0first\t\r \u00a0line\t\r \u00a0drugs)\t\r \u00a0for\t\r \u00a0over\t\r \u00a050%\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0isolates\t\r \u00a0has\t\r \u00a0been\t\r \u00a0reported\t\r \u00a0in\t\r \u00a0several\t\r \u00a0countries(5).\t\r \u00a0As\t\r \u00a0a\t\r \u00a0result,\t\r \u00a0quinolones\t\r \u00a0and\t\r \u00a0fluoroquinolones\t\r \u00a0are\t\r \u00a0now\t\r \u00a0the\t\r \u00a0most\t\r \u00a0commonly\t\r \u00a0used\t\r \u00a0antimicrobial\t\r \u00a0used\t\r \u00a0to\t\r \u00a0treat\t\r \u00a0Salmonella\t\r \u00a0infections.\t\r \u00a0However,\t\r \u00a0resistance\t\r \u00a0to\t\r \u00a0these\t\r \u00a0antimicrobials\t\r \u00a0is\t\r \u00a0increasing\t\r \u00a0as\t\r \u00a0well.\t\r \u00a0Studies\t\r \u00a0on\t\r \u00a0isolates\t\r \u00a0from\t\r \u00a0hospitalized\t\r \u00a0patients\t\r \u00a0in\t\r \u00a0Bangalore,\t\r \u00a0India\t\r \u00a0and\t\r \u00a0Lagos,\t\r \u00a0Nigeria\t\r \u00a0both\t\r \u00a0reported\t\r \u00a0over\t\r \u00a060%\t\r \u00a0resistance\t\r \u00a0to\t\r \u00a0nalidixic\t\r \u00a0acid,\t\r \u00a0one\t\r \u00a0of\t\r \u00a0the\t\r \u00a0first\t\r \u00a0developed\t\r \u00a0quinolones(6,\t\r \u00a07).\t\r \u00a0Furthermore,\t\r \u00a0a\t\r \u00a0study\t\r \u00a0in\t\r \u00a0Kathmandu,\t\r \u00a0Nepal\t\r \u00a0reported\t\r \u00a05%\t\r \u00a0resistance\t\r \u00a0of\t\r \u00a0S.\t\r \u00a0Typhi\t\r \u00a0isolates\t\r \u00a0and\t\r \u00a013%\t\r \u00a0resistance\t\r \u00a0of\t\r \u00a0S.\t\r \u00a0Paratyphi\t\r \u00a0isolates\t\r \u00a0to\t\r \u00a0ciprofloxin,\t\r \u00a0which\t\r \u00a0is\t\r \u00a0a\t\r \u00a0fluoroquinolone\t\r \u00a0drug(8).\t\r \u00a0\t\r \u00a0 \t\r \u00a0\t\r \u00a0 3\t\r \u00a0 1.1.3\t\r \u00a0Salmonella\t\r \u00a0pathogenesis\t\r \u00a0 1.1.3.1\t\r \u00a0Salmonella\t\r \u00a0pathogenesis\t\r \u00a0within\t\r \u00a0host\t\r \u00a0organisms\t\r \u00a0In\t\r \u00a0humans,\t\r \u00a0Salmonella\t\r \u00a0are\t\r \u00a0typically\t\r \u00a0acquired\t\r \u00a0through\t\r \u00a0the\t\r \u00a0ingestion\t\r \u00a0of\t\r \u00a0contaminated\t\r \u00a0food\t\r \u00a0or\t\r \u00a0water.\t\r \u00a0Most\t\r \u00a0commonly\t\r \u00a0this\t\r \u00a0consists\t\r \u00a0of\t\r \u00a0animal\t\r \u00a0products\t\r \u00a0such\t\r \u00a0as\t\r \u00a0meat,\t\r \u00a0poultry,\t\r \u00a0eggs\t\r \u00a0and\t\r \u00a0milk.\t\r \u00a0However,\t\r \u00a0other\t\r \u00a0foods\t\r \u00a0such\t\r \u00a0as\t\r \u00a0fruits\t\r \u00a0and\t\r \u00a0vegetables\t\r \u00a0grown\t\r \u00a0in\t\r \u00a0contaminated\t\r \u00a0manure\t\r \u00a0can\t\r \u00a0also\t\r \u00a0transmit\t\r \u00a0the\t\r \u00a0bacteria.\t\r \u00a0Once\t\r \u00a0ingested,\t\r \u00a0 Salmonella\t\r \u00a0enter\t\r \u00a0the\t\r \u00a0stomach,\t\r \u00a0where\t\r \u00a0an\t\r \u00a0adaptive\t\r \u00a0acid\t\r \u00a0tolerance\t\r \u00a0response\t\r \u00a0has\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0promote\t\r \u00a0their\t\r \u00a0survival\t\r \u00a0at\t\r \u00a0low\t\r \u00a0pH\t\r \u00a0in\t\r \u00a0gastric\t\r \u00a0juices(9,\t\r \u00a010).\t\r \u00a0They\t\r \u00a0then\t\r \u00a0colonize\t\r \u00a0the\t\r \u00a0ileum\t\r \u00a0of\t\r \u00a0the\t\r \u00a0small\t\r \u00a0intestine,\t\r \u00a0initially\t\r \u00a0traversing\t\r \u00a0the\t\r \u00a0intestinal\t\r \u00a0mucous\t\r \u00a0layer\t\r \u00a0to\t\r \u00a0gain\t\r \u00a0access\t\r \u00a0to\t\r \u00a0the\t\r \u00a0underlying\t\r \u00a0epithelium(11-\u00ad\u201013).\t\r \u00a0From\t\r \u00a0here,\t\r \u00a0fimbrial\t\r \u00a0adhesins\t\r \u00a0are\t\r \u00a0thought\t\r \u00a0to\t\r \u00a0mediate\t\r \u00a0binding\t\r \u00a0to\t\r \u00a0target\t\r \u00a0cells,\t\r \u00a0and\t\r \u00a0several\t\r \u00a0mechanisms\t\r \u00a0are\t\r \u00a0used\t\r \u00a0to\t\r \u00a0breach\t\r \u00a0the\t\r \u00a0host\t\r \u00a0epithelium(14,\t\r \u00a015).\t\r \u00a0Salmonella\t\r \u00a0can\t\r \u00a0induce\t\r \u00a0membrane\t\r \u00a0ruffling\t\r \u00a0and\t\r \u00a0macropinocytosis\t\r \u00a0to\t\r \u00a0actively\t\r \u00a0invade\t\r \u00a0and\t\r \u00a0transcytose\t\r \u00a0microfold\t\r \u00a0cells\t\r \u00a0(M\t\r \u00a0cells)\t\r \u00a0for\t\r \u00a0delivery\t\r \u00a0to\t\r \u00a0underlying\t\r \u00a0Peyer\u2019s\t\r \u00a0Patches(16,\t\r \u00a017).\t\r \u00a0However,\t\r \u00a0they\t\r \u00a0can\t\r \u00a0also\t\r \u00a0be\t\r \u00a0phagocytosed\t\r \u00a0by\t\r \u00a0dendritic\t\r \u00a0cells,\t\r \u00a0which\t\r \u00a0use\t\r \u00a0tight\t\r \u00a0junctions\t\r \u00a0to\t\r \u00a0access\t\r \u00a0the\t\r \u00a0epithelium\t\r \u00a0and\t\r \u00a0sample\t\r \u00a0the\t\r \u00a0intestinal\t\r \u00a0lumen(18,\t\r \u00a019).\t\r \u00a0In\t\r \u00a0vitro\t\r \u00a0Salmonella\t\r \u00a0have\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0disrupt\t\r \u00a0epithelial\t\r \u00a0tight\t\r \u00a0junctions,\t\r \u00a0which\t\r \u00a0suggests\t\r \u00a0another\t\r \u00a0mechanism\t\r \u00a0whereby\t\r \u00a0they\t\r \u00a0might\t\r \u00a0breach\t\r \u00a0the\t\r \u00a0intestinal\t\r \u00a0barrier(20).\t\r \u00a0It\t\r \u00a0is\t\r \u00a0also\t\r \u00a0possible\t\r \u00a0that,\t\r \u00a0once\t\r \u00a0in\t\r \u00a0Peyer\u2019s\t\r \u00a0patches,\t\r \u00a0Salmonella\t\r \u00a0re-\u00ad\u2010enter\t\r \u00a0enterocytes\t\r \u00a0from\t\r \u00a0the\t\r \u00a0basolateral\t\r \u00a0membrane(21).\t\r \u00a0\t\r \u00a0Once\t\r \u00a0the\t\r \u00a0intestinal\t\r \u00a0barrier\t\r \u00a0has\t\r \u00a0been\t\r \u00a0breached,\t\r \u00a0serovars\t\r \u00a0causing\t\r \u00a0gastroenteritis\t\r \u00a0induce\t\r \u00a0a\t\r \u00a0local\t\r \u00a0inflammatory\t\r \u00a0response.\t\r \u00a0This\t\r \u00a0results\t\r \u00a0in\t\r \u00a0Interleukin\t\r \u00a08\t\r \u00a0secretion\t\r \u00a0(IL-\u00ad\u20108)\t\r \u00a0by\t\r \u00a0the\t\r \u00a0infected\t\r \u00a0epithelium,\t\r \u00a0infiltration\t\r \u00a0of\t\r \u00a0destructive\t\r \u00a0polymorphonuclear\t\r \u00a0leukocytes\t\r \u00a0(PMNs),\t\r \u00a0and\t\r \u00a0a\t\r \u00a0loss\t\r \u00a0in\t\r \u00a0the\t\r \u00a0epithelial\t\r \u00a0lining\t\r \u00a0and\t\r \u00a0 \t\r \u00a0\t\r \u00a0 4\t\r \u00a0 adsorptive\t\r \u00a0surface(22).\t\r \u00a0Often\t\r \u00a0this\t\r \u00a0leads\t\r \u00a0to\t\r \u00a0fever,\t\r \u00a0abdominal\t\r \u00a0pains\t\r \u00a0and\t\r \u00a0diarrhea,\t\r \u00a0but\t\r \u00a0the\t\r \u00a0infection\t\r \u00a0does\t\r \u00a0not\t\r \u00a0spread\t\r \u00a0elsewhere\t\r \u00a0in\t\r \u00a0the\t\r \u00a0body\t\r \u00a0and\t\r \u00a0is\t\r \u00a0most\t\r \u00a0often\t\r \u00a0self\t\r \u00a0limiting.\t\r \u00a0However,\t\r \u00a0in\t\r \u00a0the\t\r \u00a0case\t\r \u00a0of\t\r \u00a0typhoid\t\r \u00a0fever,\t\r \u00a0penetration\t\r \u00a0of\t\r \u00a0the\t\r \u00a0intestinal\t\r \u00a0barrier\t\r \u00a0requires\t\r \u00a0very\t\r \u00a0few\t\r \u00a0bacteria\t\r \u00a0and\t\r \u00a0causes\t\r \u00a0little\t\r \u00a0inflammation.\t\r \u00a0Because\t\r \u00a0of\t\r \u00a0this,\t\r \u00a0it\t\r \u00a0has\t\r \u00a0been\t\r \u00a0speculated\t\r \u00a0that\t\r \u00a0typhi\t\r \u00a0and\t\r \u00a0paratyphi\t\r \u00a0serovars\t\r \u00a0may\t\r \u00a0primarily\t\r \u00a0target\t\r \u00a0dendritic\t\r \u00a0cells,\t\r \u00a0and\t\r \u00a0Salmonella\t\r \u00a0genes\t\r \u00a0required\t\r \u00a0for\t\r \u00a0epithelial\t\r \u00a0cell\t\r \u00a0invasion\t\r \u00a0have\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0be\t\r \u00a0dispensible\t\r \u00a0for\t\r \u00a0enteric\t\r \u00a0fever(23-\u00ad\u201025).\t\r \u00a0Once\t\r \u00a0across\t\r \u00a0the\t\r \u00a0intestinal\t\r \u00a0barrier,\t\r \u00a0serovars\t\r \u00a0causing\t\r \u00a0systemic\t\r \u00a0illness\t\r \u00a0enter\t\r \u00a0tissue\t\r \u00a0macrophages\t\r \u00a0where\t\r \u00a0they\t\r \u00a0have\t\r \u00a0the\t\r \u00a0distinct\t\r \u00a0ability\t\r \u00a0to\t\r \u00a0survive\t\r \u00a0and\t\r \u00a0replicate\t\r \u00a0intracellularly(26-\u00ad\u201029).\t\r \u00a0This\t\r \u00a0allows\t\r \u00a0bacterial\t\r \u00a0replication\t\r \u00a0and\t\r \u00a0subsequent\t\r \u00a0spread\t\r \u00a0throughout\t\r \u00a0the\t\r \u00a0reticuloendothelial\t\r \u00a0system\t\r \u00a0resulting\t\r \u00a0in\t\r \u00a0severe\t\r \u00a0systemic\t\r \u00a0infection.\t\r \u00a0Physiological\t\r \u00a0outcomes\t\r \u00a0of\t\r \u00a0typhoid\t\r \u00a0fever\t\r \u00a0include\t\r \u00a0hepatosplenomegaly\t\r \u00a0and\t\r \u00a0sustained\t\r \u00a0fevers\t\r \u00a0of\t\r \u00a0temperatures\t\r \u00a0between\t\r \u00a039-\u00ad\u201040\u00b0C(30).\t\r \u00a0\t\r \u00a0 1.1.3.2\t\r \u00a0Cellular\t\r \u00a0aspects\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0pathogenesis\t\r \u00a0 1.1.3.2.1\t\r \u00a0Salmonella-\u00ad\u2010induced\t\r \u00a0signaling\t\r \u00a0cascades\t\r \u00a0Several\t\r \u00a0innate\t\r \u00a0immune\t\r \u00a0signaling\t\r \u00a0cascades\t\r \u00a0are\t\r \u00a0activated\t\r \u00a0as\t\r \u00a0Salmonella\t\r \u00a0invade\t\r \u00a0epithelial\t\r \u00a0cells.\t\r \u00a0These\t\r \u00a0are\t\r \u00a0initiated\t\r \u00a0by\t\r \u00a0Cell\t\r \u00a0division\t\r \u00a0control\t\r \u00a0protein\t\r \u00a042\t\r \u00a0(Cdc42)\t\r \u00a0and\t\r \u00a0Protein\t\r \u00a0kinase\t\r \u00a0B\t\r \u00a0(Akt)\t\r \u00a0activation,\t\r \u00a0and\t\r \u00a0lead\t\r \u00a0to\t\r \u00a0signaling\t\r \u00a0through\t\r \u00a0the\t\r \u00a0Extracellular\t\r \u00a0signal-\u00ad\u2010regulated\t\r \u00a0kinase\t\r \u00a0(ERK)\t\r \u00a01\t\r \u00a0and\t\r \u00a02,\t\r \u00a0c-\u00ad\u2010Jun\t\r \u00a0N-\u00ad\u2010terminal\t\r \u00a0kinase\t\r \u00a0(JNK),\t\r \u00a0and\t\r \u00a0Mitogen-\u00ad\u2010activated\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a0p38\t\r \u00a0(p38)\t\r \u00a0signaling\t\r \u00a0cascades\t\r \u00a0resulting\t\r \u00a0in\t\r \u00a0the\t\r \u00a0production\t\r \u00a0of\t\r \u00a0proinflammatory\t\r \u00a0cytokines\t\r \u00a0such\t\r \u00a0as\t\r \u00a0IL-\u00ad\u20108(31-\u00ad\u201033).\t\r \u00a0Furthermore,\t\r \u00a0epithelial\t\r \u00a0cells\t\r \u00a0undergo\t\r \u00a0apoptosis\t\r \u00a012-\u00ad\u201018\t\r \u00a0hours\t\r \u00a0(h)\t\r \u00a0post\t\r \u00a0invasion\t\r \u00a0by\t\r \u00a0Salmonella,\t\r \u00a0which\t\r \u00a0results\t\r \u00a0in\t\r \u00a0 \t\r \u00a0\t\r \u00a0 5\t\r \u00a0 activation\t\r \u00a0of\t\r \u00a0caspase-\u00ad\u20103\t\r \u00a0and\t\r \u00a0caspase-\u00ad\u20108,\t\r \u00a0but\t\r \u00a0not\t\r \u00a0caspase-\u00ad\u20101(34-\u00ad\u201036).\t\r \u00a0Alternatively,\t\r \u00a0macrophages\t\r \u00a0and\t\r \u00a0dendritic\t\r \u00a0cells\t\r \u00a0exposed\t\r \u00a0to\t\r \u00a0invasive\t\r \u00a0Salmonella\t\r \u00a0can\t\r \u00a0die\t\r \u00a0within\t\r \u00a01\t\r \u00a0h\t\r \u00a0post\t\r \u00a0infection(37-\u00ad\u201039).\t\r \u00a0This\t\r \u00a0is\t\r \u00a0dependent\t\r \u00a0on\t\r \u00a0caspase-\u00ad\u20101,\t\r \u00a0which\t\r \u00a0is\t\r \u00a0activated\t\r \u00a0by\t\r \u00a0Nucleotide\t\r \u00a0oligomerization\t\r \u00a0domain-\u00ad\u2010like\t\r \u00a0receptors\t\r \u00a0(NLRs)\t\r \u00a0within\t\r \u00a0inflammasomes,\t\r \u00a0and,\t\r \u00a0distinct\t\r \u00a0from\t\r \u00a0apoptosis,\t\r \u00a0is\t\r \u00a0proinflammatory\t\r \u00a0and\t\r \u00a0results\t\r \u00a0in\t\r \u00a0rapid\t\r \u00a0cell\t\r \u00a0lysis(37,\t\r \u00a040-\u00ad\u201042).\t\r \u00a0\t\r \u00a0 1.1.3.2.2\t\r \u00a0Internalization\t\r \u00a0into\t\r \u00a0the\t\r \u00a0Salmonella-\u00ad\u2010containing\t\r \u00a0vacuole\t\r \u00a0 Salmonella\t\r \u00a0actively\t\r \u00a0enter\t\r \u00a0epithelial\t\r \u00a0cells\t\r \u00a0through\t\r \u00a0dramatic\t\r \u00a0remodeling\t\r \u00a0of\t\r \u00a0the\t\r \u00a0actin\t\r \u00a0cytoskeleton,\t\r \u00a0the\t\r \u00a0formation\t\r \u00a0of\t\r \u00a0membrane\t\r \u00a0ruffles\t\r \u00a0at\t\r \u00a0the\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0surface,\t\r \u00a0and\t\r \u00a0are\t\r \u00a0internalized\t\r \u00a0into\t\r \u00a0a\t\r \u00a0membrane\t\r \u00a0bound\t\r \u00a0compartment\t\r \u00a0termed\t\r \u00a0the\t\r \u00a0Salmonella-\u00ad\u2010containing\t\r \u00a0vacuole\t\r \u00a0(SCV)\t\r \u00a0(Figure\t\r \u00a01.1).\t\r \u00a0These\t\r \u00a0bacterial\t\r \u00a0entry\t\r \u00a0sites\t\r \u00a0are\t\r \u00a0enriched\t\r \u00a0in\t\r \u00a0cholesterol,\t\r \u00a0which\t\r \u00a0is\t\r \u00a0essential\t\r \u00a0for\t\r \u00a0internalization\t\r \u00a0of\t\r \u00a0the\t\r \u00a0bacteria,\t\r \u00a0and\t\r \u00a0aggregation\t\r \u00a0of\t\r \u00a0specific\t\r \u00a0cell\t\r \u00a0surface\t\r \u00a0proteins\t\r \u00a0has\t\r \u00a0been\t\r \u00a0observed(43,\t\r \u00a044).\t\r \u00a0Membrane\t\r \u00a0ruffling\t\r \u00a0involves\t\r \u00a0the\t\r \u00a0small\t\r \u00a0guanosine\t\r \u00a0triphosphatases\t\r \u00a0(GTPases)\t\r \u00a0Ras-\u00ad\u2010related\t\r \u00a0C3\t\r \u00a0bolulinum\t\r \u00a0toxin\t\r \u00a0substrate\t\r \u00a01\t\r \u00a0(Rac1)\t\r \u00a0and\t\r \u00a0Rho-\u00ad\u2010related\t\r \u00a0GTP-\u00ad\u2010binding\t\r \u00a0protein\t\r \u00a0RhoG\t\r \u00a0(RhoG),\t\r \u00a0which\t\r \u00a0activate\t\r \u00a0Wiskott-\u00ad\u2010Aldrich\t\r \u00a0syndrome\t\r \u00a0protein\t\r \u00a0(WASP)\t\r \u00a0family\t\r \u00a0members\t\r \u00a0and\t\r \u00a0recruit\t\r \u00a0the\t\r \u00a0Actin-\u00ad\u2010related\t\r \u00a0protein-\u00ad\u20102\/3\t\r \u00a0(Arp2\/3)\t\r \u00a0complex\t\r \u00a0to\t\r \u00a0stimulate\t\r \u00a0actin\t\r \u00a0polymerization(45-\u00ad\u201048).\t\r \u00a0Furthermore,\t\r \u00a0invaginations\t\r \u00a0at\t\r \u00a0the\t\r \u00a0base\t\r \u00a0of\t\r \u00a0membrane\t\r \u00a0ruffles\t\r \u00a0are\t\r \u00a0depleted\t\r \u00a0for\t\r \u00a0Phosphatidylinositol\t\r \u00a04,5-\u00ad\u2010bisphosphate\t\r \u00a0(PI(4,5)P2),\t\r \u00a0which\t\r \u00a0likely\t\r \u00a0causes\t\r \u00a0dissociation\t\r \u00a0of\t\r \u00a0actin\t\r \u00a0and\t\r \u00a0membrane\t\r \u00a0proteins\t\r \u00a0to\t\r \u00a0promote\t\r \u00a0fission\t\r \u00a0of\t\r \u00a0vacuole(49).\t\r \u00a0Vesicle-\u00ad\u2010associated\t\r \u00a0membrane\t\r \u00a0protein\t\r \u00a08\t\r \u00a0(VAMP8)\t\r \u00a0positive\t\r \u00a0vesicles\t\r \u00a0are\t\r \u00a0also\t\r \u00a0recruited\t\r \u00a0to\t\r \u00a0and\t\r \u00a0 \t\r \u00a0\t\r \u00a0 6\t\r \u00a0 fuse\t\r \u00a0with\t\r \u00a0these\t\r \u00a0ruffles,\t\r \u00a0providing\t\r \u00a0a\t\r \u00a0membrane\t\r \u00a0source\t\r \u00a0for\t\r \u00a0the\t\r \u00a0forming\t\r \u00a0macropinosome(50).\t\r \u00a0 Figure\t\r \u00a01.1\t\r \u00a0Diagram\t\r \u00a0of\t\r \u00a0SCV\t\r \u00a0biogenesis\t\r \u00a0and\t\r \u00a0maturation\t\r \u00a0 \t\r \u00a0Host\t\r \u00a0actin\t\r \u00a0dynamics\t\r \u00a0and\t\r \u00a0membrane\t\r \u00a0ruffling\t\r \u00a0are\t\r \u00a0depicted,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0interactions\t\r \u00a0with\t\r \u00a0early\t\r \u00a0and\t\r \u00a0late\t\r \u00a0endosomes,\t\r \u00a0and\t\r \u00a0to\t\r \u00a0a\t\r \u00a0currently\t\r \u00a0unknown\t\r \u00a0extent,\t\r \u00a0lysosomes.\t\r \u00a0The\t\r \u00a0formation\t\r \u00a0of\t\r \u00a0SIFs\t\r \u00a0along\t\r \u00a0microtubules\t\r \u00a0is\t\r \u00a0represented,\t\r \u00a0and\t\r \u00a0Salmonella\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0are\t\r \u00a0shown\t\r \u00a0in\t\r \u00a0blue,\t\r \u00a0while\t\r \u00a0T3SS-\u00ad\u20102\t\r \u00a0is\t\r \u00a0shown\t\r \u00a0in\t\r \u00a0red.\t\r \u00a0\t\r \u00a0 1.1.3.2.3\t\r \u00a0Maturation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV\t\r \u00a0Following\t\r \u00a0invasion,\t\r \u00a0the\t\r \u00a0SCV\t\r \u00a0undergoes\t\r \u00a0an\t\r \u00a0initial\t\r \u00a0maturation\t\r \u00a0process\t\r \u00a0similar\t\r \u00a0to\t\r \u00a0nascent\t\r \u00a0phagosomes(51-\u00ad\u201053).\t\r \u00a0Initially,\t\r \u00a0the\t\r \u00a0vacuole\t\r \u00a0transiently\t\r \u00a0acquires\t\r \u00a0early\t\r \u00a0endosome\t\r \u00a0markers\t\r \u00a0such\t\r \u00a0as\t\r \u00a0transferrin\t\r \u00a0receptor,\t\r \u00a0early\t\r \u00a0endosome\t\r \u00a0antigen\t\r \u00a01\t\r \u00a0and\t\r \u00a0Ras-\u00ad\u2010related\t\r \u00a0protein\t\r \u00a0Rab5\t\r \u00a0(Rab5),\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0Phosphatidylinositol\t\r \u00a03-\u00ad\u2010kinase\t\r \u00a0catalytic\t\r \u00a0subunit\t\r \u00a0type\t\r \u00a03\t\r \u00a0(Vps34)\t\r \u00a0which\t\r \u00a0generates\t\r \u00a0high\t\r \u00a0levels\t\r \u00a0of\t\r \u00a0Phosphatidylinositol\t\r \u00a03-\u00ad\u2010phosphate\t\r \u00a0(PI3P)\t\r \u00a0on\t\r \u00a0the\t\r \u00a0SCV(54-\u00ad\u201058).\t\r \u00a0Sorting\t\r \u00a0nexins-\u00ad\u20101\t\r \u00a0and\t\r \u00a0-\u00ad\u20103\t\r \u00a0(SNX1\t\r \u00a0and\t\r \u00a0SNX3),\t\r \u00a0 \t\r \u00a0\t\r \u00a0 7\t\r \u00a0 which\t\r \u00a0bind\t\r \u00a0phosphoinositides,\t\r \u00a0are\t\r \u00a0also\t\r \u00a0recruited\t\r \u00a0transiently\t\r \u00a0to\t\r \u00a0the\t\r \u00a0early\t\r \u00a0SCV.\t\r \u00a0Both\t\r \u00a0result\t\r \u00a0in\t\r \u00a0the\t\r \u00a0formation\t\r \u00a0of\t\r \u00a0long\t\r \u00a0spacious\t\r \u00a0vacuolar-\u00ad\u2010associated\t\r \u00a0tubules,\t\r \u00a0which\t\r \u00a0extend\t\r \u00a0from\t\r \u00a0large\t\r \u00a0macropinosomes\t\r \u00a0reducing\t\r \u00a0their\t\r \u00a0size(59,\t\r \u00a060).\t\r \u00a0SNX3\t\r \u00a0has\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0bind\t\r \u00a0PI3P\t\r \u00a0on\t\r \u00a0SCVs,\t\r \u00a0and\t\r \u00a0SNX1\t\r \u00a0regulates\t\r \u00a0retrograde\t\r \u00a0transport\t\r \u00a0to\t\r \u00a0the\t\r \u00a0trans-\u00ad\u2010Golgi\t\r \u00a0network\t\r \u00a0(TGN),\t\r \u00a0consistent\t\r \u00a0with\t\r \u00a0reports\t\r \u00a0that\t\r \u00a0the\t\r \u00a0early\t\r \u00a0SCV\t\r \u00a0interacts\t\r \u00a0extensively\t\r \u00a0with\t\r \u00a0the\t\r \u00a0endocytic\t\r \u00a0recycling\t\r \u00a0system(61).\t\r \u00a0Following\t\r \u00a0the\t\r \u00a0initial\t\r \u00a0acquisition\t\r \u00a0of\t\r \u00a0early\t\r \u00a0endocytic\t\r \u00a0markers,\t\r \u00a0the\t\r \u00a0SCV\t\r \u00a0also\t\r \u00a0acquires\t\r \u00a0several\t\r \u00a0markers\t\r \u00a0of\t\r \u00a0late\t\r \u00a0endosomes\t\r \u00a0and\t\r \u00a0lysosomes\t\r \u00a0such\t\r \u00a0as\t\r \u00a0Rab7,\t\r \u00a0Lysosomal-\u00ad\u2010associated\t\r \u00a0membrane\t\r \u00a0proteins\t\r \u00a0(LAMPs)\t\r \u00a01\t\r \u00a0and\t\r \u00a02,\t\r \u00a0and\t\r \u00a0Vacuolar-\u00ad\u2010type\t\r \u00a0H+-\u00ad\u2010ATPase\t\r \u00a0(v-\u00ad\u2010ATPase)(54,\t\r \u00a062).\t\r \u00a0However,\t\r \u00a0neither\t\r \u00a0Cation-\u00ad\u2010dependent\t\r \u00a0nor\t\r \u00a0Cation\u2013independent\t\r \u00a0mannose-\u00ad\u20106-\u00ad\u2010phosphate\t\r \u00a0receptors\t\r \u00a0(M6PRs)\t\r \u00a0have\t\r \u00a0been\t\r \u00a0identified\t\r \u00a0on\t\r \u00a0the\t\r \u00a0SCV,\t\r \u00a0and\t\r \u00a0only\t\r \u00a0small\t\r \u00a0amounts\t\r \u00a0of\t\r \u00a0hydrolases\t\r \u00a0such\t\r \u00a0as\t\r \u00a0Cathepsin\t\r \u00a0D\t\r \u00a0have\t\r \u00a0been\t\r \u00a0reported(54,\t\r \u00a062).\t\r \u00a0Thus,\t\r \u00a0it\t\r \u00a0has\t\r \u00a0been\t\r \u00a0proposed\t\r \u00a0that\t\r \u00a0the\t\r \u00a0SCV\t\r \u00a0continuously\t\r \u00a0interacts\t\r \u00a0with\t\r \u00a0only\t\r \u00a0a\t\r \u00a0specific\t\r \u00a0subset\t\r \u00a0of\t\r \u00a0a\t\r \u00a0very\t\r \u00a0heterogeneous\t\r \u00a0population\t\r \u00a0of\t\r \u00a0late\t\r \u00a0endosomes\t\r \u00a0and\t\r \u00a0lysosomes(44).\t\r \u00a0Consistent\t\r \u00a0with\t\r \u00a0this,\t\r \u00a0several\t\r \u00a0Rab7\t\r \u00a0positive\t\r \u00a0compartments\t\r \u00a0rich\t\r \u00a0in\t\r \u00a0LAMPs\t\r \u00a0but\t\r \u00a0containing\t\r \u00a0only\t\r \u00a0very\t\r \u00a0low\t\r \u00a0levels\t\r \u00a0of\t\r \u00a0Cathepsin\t\r \u00a0D\t\r \u00a0have\t\r \u00a0been\t\r \u00a0show\t\r \u00a0to\t\r \u00a0accumulate\t\r \u00a0around\t\r \u00a0the\t\r \u00a0SCV(63).\t\r \u00a0However,\t\r \u00a0it\t\r \u00a0is\t\r \u00a0also\t\r \u00a0possible\t\r \u00a0that\t\r \u00a0SNX1\t\r \u00a0dependent\t\r \u00a0removal\t\r \u00a0of\t\r \u00a0Golgi\t\r \u00a0cargo\t\r \u00a0also\t\r \u00a0depletes\t\r \u00a0M6PRs\t\r \u00a0from\t\r \u00a0the\t\r \u00a0SCV(59).\t\r \u00a0How\t\r \u00a0the\t\r \u00a0SCV\t\r \u00a0selects\t\r \u00a0and\t\r \u00a0interacts\t\r \u00a0with\t\r \u00a0specific\t\r \u00a0endosomal\t\r \u00a0compartments\t\r \u00a0is\t\r \u00a0largely\t\r \u00a0unknown.\t\r \u00a0However,\t\r \u00a0acquisition\t\r \u00a0of\t\r \u00a0some\t\r \u00a0host\t\r \u00a0trafficking\t\r \u00a0proteins\t\r \u00a0is\t\r \u00a0known\t\r \u00a0to\t\r \u00a0be\t\r \u00a0essential\t\r \u00a0for\t\r \u00a0SCV\t\r \u00a0maturation.\t\r \u00a0Inhibition\t\r \u00a0of\t\r \u00a01-\u00ad\u2010phosphatidylinositol-\u00ad\u20103-\u00ad\u2010phosphate\t\r \u00a05-\u00ad\u2010kinase\t\r \u00a0(PIKFYVE),\t\r \u00a0which\t\r \u00a0regulates\t\r \u00a0fusion\t\r \u00a0of\t\r \u00a0early\t\r \u00a0endosomes\t\r \u00a0with\t\r \u00a0late\t\r \u00a0endosomes\/lysosomal\t\r \u00a0compartments,\t\r \u00a0was\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0block\t\r \u00a0acidification\t\r \u00a0of,\t\r \u00a0and\t\r \u00a0 \t\r \u00a0\t\r \u00a0 8\t\r \u00a0 LAMP1\t\r \u00a0accumulation\t\r \u00a0on,\t\r \u00a0the\t\r \u00a0SCV(64).\t\r \u00a0Furthermore,\t\r \u00a0several\t\r \u00a0Rab\t\r \u00a0GTPases\t\r \u00a0are\t\r \u00a0recruited\t\r \u00a0differently\t\r \u00a0to\t\r \u00a0model\t\r \u00a0phagosomes\t\r \u00a0as\t\r \u00a0compared\t\r \u00a0to\t\r \u00a0SCVs.\t\r \u00a0Isoforms\t\r \u00a0of\t\r \u00a0Rab5\t\r \u00a0and\t\r \u00a0Rab11\t\r \u00a0are\t\r \u00a0present\t\r \u00a0in\t\r \u00a0much\t\r \u00a0higher\t\r \u00a0levels\t\r \u00a0on\t\r \u00a0SCVs\t\r \u00a0as\t\r \u00a0compared\t\r \u00a0to\t\r \u00a0model\t\r \u00a0phagosomes(65).\t\r \u00a0Interestingly,\t\r \u00a0Rab11\t\r \u00a0is\t\r \u00a0implicated\t\r \u00a0in\t\r \u00a0endosomal\t\r \u00a0recycling,\t\r \u00a0consistent\t\r \u00a0with\t\r \u00a0extensive\t\r \u00a0interactions\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV\t\r \u00a0with\t\r \u00a0recycling\t\r \u00a0endosomes.\t\r \u00a0Rab7,\t\r \u00a0which\t\r \u00a0is\t\r \u00a0required\t\r \u00a0for\t\r \u00a0LAMP1\t\r \u00a0accumulation\t\r \u00a0on\t\r \u00a0the\t\r \u00a0SCV,\t\r \u00a0is\t\r \u00a0also\t\r \u00a0present\t\r \u00a0on\t\r \u00a0SCVs\t\r \u00a0much\t\r \u00a0earlier\t\r \u00a0than\t\r \u00a0model\t\r \u00a0phagosomes(63,\t\r \u00a065).\t\r \u00a0Interestingly,\t\r \u00a0Rab\t\r \u00a08B,\t\r \u00a013,\t\r \u00a023\t\r \u00a0and\t\r \u00a035\t\r \u00a0are\t\r \u00a0found\t\r \u00a0on\t\r \u00a0model\t\r \u00a0phagosomes\t\r \u00a0but\t\r \u00a0not\t\r \u00a0SCVs,\t\r \u00a0and\t\r \u00a0Rab\t\r \u00a023\t\r \u00a0and\t\r \u00a035\t\r \u00a0have\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0regulate\t\r \u00a0phagosome-\u00ad\u2010lysosome\t\r \u00a0fusion(65).\t\r \u00a0Inactivation\t\r \u00a0of\t\r \u00a0Rab14\t\r \u00a0by\t\r \u00a0AS160\t\r \u00a0has\t\r \u00a0also\t\r \u00a0been\t\r \u00a0proposed\t\r \u00a0to\t\r \u00a0prevent\t\r \u00a0maturation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV(66).\t\r \u00a0\t\r \u00a0 1.1.3.2.4\t\r \u00a0Salmonella\u2019s\t\r \u00a0replicative\t\r \u00a0niche\t\r \u00a0Several\t\r \u00a0hours\t\r \u00a0post\t\r \u00a0invasion,\t\r \u00a0the\t\r \u00a0SCV\t\r \u00a0adopts\t\r \u00a0a\t\r \u00a0perinuclear\t\r \u00a0localization\t\r \u00a0where\t\r \u00a0the\t\r \u00a0bacteria\t\r \u00a0establish\t\r \u00a0a\t\r \u00a0replicative\t\r \u00a0niche\t\r \u00a0within\t\r \u00a0the\t\r \u00a0host\t\r \u00a0cell.\t\r \u00a0Approximately\t\r \u00a030\t\r \u00a0minutes\t\r \u00a0(min)\t\r \u00a0post\t\r \u00a0invasion,\t\r \u00a0Rab7\t\r \u00a0recruits\t\r \u00a0Rab7-\u00ad\u2010interacting\t\r \u00a0lysosomal\t\r \u00a0protein\t\r \u00a0(RILP)\t\r \u00a0to\t\r \u00a0the\t\r \u00a0SCV,\t\r \u00a0which\t\r \u00a0recruits\t\r \u00a0dynein,\t\r \u00a0resulting\t\r \u00a0in\t\r \u00a0movement\t\r \u00a0along\t\r \u00a0microtubules\t\r \u00a0and\t\r \u00a0towards\t\r \u00a0a\t\r \u00a0perinuclear\t\r \u00a0localization(67-\u00ad\u201069).\t\r \u00a0However,\t\r \u00a0movement\t\r \u00a0along\t\r \u00a0actin\t\r \u00a0filaments\t\r \u00a0is\t\r \u00a0also\t\r \u00a0thought\t\r \u00a0to\t\r \u00a0direct\t\r \u00a0positioning\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV,\t\r \u00a0as\t\r \u00a0activation\t\r \u00a0of\t\r \u00a0Myosin\t\r \u00a0II\t\r \u00a0downstream\t\r \u00a0of\t\r \u00a0Rho-\u00ad\u2010GTPases\t\r \u00a0and\t\r \u00a0Rho-\u00ad\u2010kinase\t\r \u00a0is\t\r \u00a0also\t\r \u00a0required\t\r \u00a0for\t\r \u00a0positioning\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV(70).\t\r \u00a0Approximately\t\r \u00a04\t\r \u00a0to\t\r \u00a06\t\r \u00a0h\t\r \u00a0post\t\r \u00a0invasion,\t\r \u00a0long\t\r \u00a0tubular\t\r \u00a0structures\t\r \u00a0termed\t\r \u00a0 Salmonella-\u00ad\u2010induced\t\r \u00a0filaments\t\r \u00a0(SIFs)\t\r \u00a0extend\t\r \u00a0from\t\r \u00a0the\t\r \u00a0SCV(71).\t\r \u00a0These\t\r \u00a0structures\t\r \u00a0are\t\r \u00a0primarily\t\r \u00a0studied\t\r \u00a0in\t\r \u00a0epithelial\t\r \u00a0cells,\t\r \u00a0but\t\r \u00a0have\t\r \u00a0also\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0in\t\r \u00a0other\t\r \u00a0cell\t\r \u00a0types\t\r \u00a0 \t\r \u00a0\t\r \u00a0 9\t\r \u00a0 such\t\r \u00a0as\t\r \u00a0macrophages(72).\t\r \u00a0Initially\t\r \u00a0SIFs\t\r \u00a0are\t\r \u00a0dynamic,\t\r \u00a0extending\t\r \u00a0and\t\r \u00a0branching\t\r \u00a0rapidly\t\r \u00a0within\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0before,\t\r \u00a0at\t\r \u00a0approximately\t\r \u00a08\t\r \u00a0h\t\r \u00a0post\t\r \u00a0infection,\t\r \u00a0they\t\r \u00a0form\t\r \u00a0extensive\t\r \u00a0but\t\r \u00a0relatively\t\r \u00a0stable\t\r \u00a0networks(73,\t\r \u00a074).\t\r \u00a0SIFs\t\r \u00a0are\t\r \u00a0required\t\r \u00a0for\t\r \u00a0bacterial\t\r \u00a0replication,\t\r \u00a0which\t\r \u00a0initiates\t\r \u00a0at\t\r \u00a0a\t\r \u00a0similar\t\r \u00a0time\t\r \u00a0post\t\r \u00a0infection,\t\r \u00a0and\t\r \u00a0have\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0contain\t\r \u00a0LAMPs,\t\r \u00a0v-\u00ad\u2010ATPase,\t\r \u00a0lysobisphosphatidic\t\r \u00a0acid\t\r \u00a0and\t\r \u00a0Rab7,\t\r \u00a0the\t\r \u00a0later\t\r \u00a0of\t\r \u00a0which\t\r \u00a0is\t\r \u00a0required\t\r \u00a0for\t\r \u00a0their\t\r \u00a0formation(75,\t\r \u00a076).\t\r \u00a0SIFs\t\r \u00a0have\t\r \u00a0been\t\r \u00a0observed\t\r \u00a0to\t\r \u00a0fuse\t\r \u00a0with\t\r \u00a0endocytic\t\r \u00a0vesicles,\t\r \u00a0and\t\r \u00a0to\t\r \u00a0contain\t\r \u00a0Secretary\t\r \u00a0carrier-\u00ad\u2010associated\t\r \u00a0membrane\t\r \u00a0protein\t\r \u00a03\t\r \u00a0(SCAMP3)(76,\t\r \u00a077).\t\r \u00a0As\t\r \u00a0SCAMPs\t\r \u00a0localize\t\r \u00a0to\t\r \u00a0the\t\r \u00a0TGN\t\r \u00a0in\t\r \u00a0uninfected\t\r \u00a0cells,\t\r \u00a0it\t\r \u00a0has\t\r \u00a0been\t\r \u00a0proposed\t\r \u00a0that\t\r \u00a0SIFs\t\r \u00a0manipulate\t\r \u00a0post-\u00ad\u2010Golgi\t\r \u00a0trafficking\t\r \u00a0to\t\r \u00a0acquire\t\r \u00a0nutrients\t\r \u00a0and\t\r \u00a0or\t\r \u00a0membrane\t\r \u00a0within\t\r \u00a0host\t\r \u00a0cells.\t\r \u00a0Consistent\t\r \u00a0with\t\r \u00a0this\t\r \u00a0view,\t\r \u00a0accumulation\t\r \u00a0of\t\r \u00a0vesicular\t\r \u00a0stomatitis\t\r \u00a0virus\t\r \u00a0glycoprotein,\t\r \u00a0which\t\r \u00a0traffics\t\r \u00a0within\t\r \u00a0exocytic\t\r \u00a0vesicles\t\r \u00a0from\t\r \u00a0the\t\r \u00a0TGN\t\r \u00a0to\t\r \u00a0the\t\r \u00a0plasma\t\r \u00a0membrane\t\r \u00a0was\t\r \u00a0observed\t\r \u00a0around\t\r \u00a0the\t\r \u00a0SCV,\t\r \u00a0and\t\r \u00a0Rab9,\t\r \u00a0which\t\r \u00a0is\t\r \u00a0involved\t\r \u00a0in\t\r \u00a0transport\t\r \u00a0between\t\r \u00a0late\t\r \u00a0endosomes\t\r \u00a0and\t\r \u00a0the\t\r \u00a0Golgi,\t\r \u00a0was\t\r \u00a0found\t\r \u00a0on\t\r \u00a0SCVs\t\r \u00a0and\t\r \u00a0SIFs\t\r \u00a0and\t\r \u00a0is\t\r \u00a0required\t\r \u00a0for\t\r \u00a0SIF\t\r \u00a0formation(65,\t\r \u00a078,\t\r \u00a079).\t\r \u00a0However,\t\r \u00a0in\t\r \u00a0contrast\t\r \u00a0to\t\r \u00a0early\t\r \u00a0trafficking\t\r \u00a0events,\t\r \u00a0dynein\t\r \u00a0is\t\r \u00a0excluded\t\r \u00a0from\t\r \u00a0SCVs\t\r \u00a0and\t\r \u00a0SIFs\t\r \u00a0at\t\r \u00a0later\t\r \u00a0time\t\r \u00a0points,\t\r \u00a0while\t\r \u00a0recruitment\t\r \u00a0of\t\r \u00a0kinesin\t\r \u00a0has\t\r \u00a0been\t\r \u00a0shown(68,\t\r \u00a080).\t\r \u00a0Throughout\t\r \u00a0maturation\t\r \u00a0and\t\r \u00a0especially\t\r \u00a0at\t\r \u00a0later\t\r \u00a0time\t\r \u00a0points\t\r \u00a0when\t\r \u00a0SIFs\t\r \u00a0are\t\r \u00a0formed,\t\r \u00a0the\t\r \u00a0SCV\t\r \u00a0is\t\r \u00a0enriched\t\r \u00a0in\t\r \u00a0cholesterol\t\r \u00a0and\t\r \u00a0a\t\r \u00a0network\t\r \u00a0of\t\r \u00a0actin\t\r \u00a0forms\t\r \u00a0around\t\r \u00a0the\t\r \u00a0vacuole.\t\r \u00a0At\t\r \u00a0early\t\r \u00a0times\t\r \u00a0post\t\r \u00a0infection\t\r \u00a0(~30\t\r \u00a0min),\t\r \u00a0cholesterol\t\r \u00a0accumulates\t\r \u00a0around\t\r \u00a0sites\t\r \u00a0of\t\r \u00a0bacterial\t\r \u00a0entry,\t\r \u00a0while\t\r \u00a0at\t\r \u00a0later\t\r \u00a0times\t\r \u00a0(~6\t\r \u00a0h),\t\r \u00a0as\t\r \u00a0much\t\r \u00a0as\t\r \u00a030%\t\r \u00a0of\t\r \u00a0total\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0cholesterol\t\r \u00a0is\t\r \u00a0on\t\r \u00a0SCVs(76,\t\r \u00a081).\t\r \u00a0This\t\r \u00a0is\t\r \u00a0striking,\t\r \u00a0as\t\r \u00a090%\t\r \u00a0of\t\r \u00a0cellular\t\r \u00a0cholesterol\t\r \u00a0is\t\r \u00a0typically\t\r \u00a0localized\t\r \u00a0to\t\r \u00a0the\t\r \u00a0plasma\t\r \u00a0membrane,\t\r \u00a0and\t\r \u00a0blocking\t\r \u00a0the\t\r \u00a0first\t\r \u00a0step\t\r \u00a0in\t\r \u00a0cholesterol\t\r \u00a0biosynthesis\t\r \u00a0inhibits\t\r \u00a0bacterial\t\r \u00a0growth\t\r \u00a0within\t\r \u00a0host\t\r \u00a0cells(82).\t\r \u00a0Around\t\r \u00a0the\t\r \u00a0 \t\r \u00a0\t\r \u00a0 10\t\r \u00a0 same\t\r \u00a0time,\t\r \u00a0F-\u00ad\u2010actin\t\r \u00a0begins\t\r \u00a0to\t\r \u00a0form\t\r \u00a0around\t\r \u00a0the\t\r \u00a0SCV,\t\r \u00a0which,\t\r \u00a0by\t\r \u00a08\t\r \u00a0h\t\r \u00a0post\t\r \u00a0infection,\t\r \u00a0forms\t\r \u00a0a\t\r \u00a0nest\t\r \u00a0around\t\r \u00a0replicating\t\r \u00a0bacteria(83).\t\r \u00a0This\t\r \u00a0is\t\r \u00a0referred\t\r \u00a0to\t\r \u00a0as\t\r \u00a0vacuole-\u00ad\u2010associated\t\r \u00a0actin\t\r \u00a0polymerization\t\r \u00a0(VAP),\t\r \u00a0and\t\r \u00a0is\t\r \u00a0important\t\r \u00a0for\t\r \u00a0Salmonella\t\r \u00a0growth\t\r \u00a0and\t\r \u00a0replication,\t\r \u00a0as\t\r \u00a0treatment\t\r \u00a0with\t\r \u00a0actin\t\r \u00a0depolymerizing\t\r \u00a0reagents\t\r \u00a0inhibits\t\r \u00a0replication\t\r \u00a0and\t\r \u00a0causes\t\r \u00a0release\t\r \u00a0of\t\r \u00a0bacteria\t\r \u00a0into\t\r \u00a0the\t\r \u00a0host\t\r \u00a0cytoplasm.\t\r \u00a0\t\r \u00a0 1.1.4\t\r \u00a0Genetics\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0virulence\t\r \u00a0At\t\r \u00a0least\t\r \u00a060\t\r \u00a0genes\t\r \u00a0are\t\r \u00a0required\t\r \u00a0for\t\r \u00a0virulence\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0enterica.\t\r \u00a0Similar\t\r \u00a0to\t\r \u00a0several\t\r \u00a0other\t\r \u00a0bacterial\t\r \u00a0pathogens,\t\r \u00a0they\t\r \u00a0are\t\r \u00a0thought\t\r \u00a0to\t\r \u00a0have\t\r \u00a0been\t\r \u00a0horizontally\t\r \u00a0acquired\t\r \u00a0and\t\r \u00a0act\t\r \u00a0in\t\r \u00a0combination\t\r \u00a0with\t\r \u00a0pre-\u00ad\u2010existing\t\r \u00a0genes\t\r \u00a0to\t\r \u00a0cause\t\r \u00a0disease(84,\t\r \u00a085).\t\r \u00a0 Salmonella\t\r \u00a0virulence\t\r \u00a0genes\t\r \u00a0are\t\r \u00a0located\t\r \u00a0on\t\r \u00a0two\t\r \u00a0pathogenicity\t\r \u00a0islands,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0a\t\r \u00a0number\t\r \u00a0of\t\r \u00a0smaller\t\r \u00a0islets\t\r \u00a0scattered\t\r \u00a0throughout\t\r \u00a0the\t\r \u00a0bacterial\t\r \u00a0chromosome.\t\r \u00a0In\t\r \u00a0addition,\t\r \u00a0several\t\r \u00a0Salmonella\t\r \u00a0serovars\t\r \u00a0also\t\r \u00a0contain\t\r \u00a0a\t\r \u00a0virulence\t\r \u00a0plasmid,\t\r \u00a0which\t\r \u00a0harbors\t\r \u00a0an\t\r \u00a0additional\t\r \u00a0pathogenicity\t\r \u00a0islet(85-\u00ad\u201087).\t\r \u00a0 Salmonella\t\r \u00a0enterica\t\r \u00a0contains\t\r \u00a0two\t\r \u00a0large\t\r \u00a0pathogenicity\t\r \u00a0islands\t\r \u00a0that\t\r \u00a0are\t\r \u00a0required\t\r \u00a0for\t\r \u00a0virulence.\t\r \u00a0Salmonella\t\r \u00a0pathogenicity\t\r \u00a0island\t\r \u00a01\t\r \u00a0(SPI-\u00ad\u20101)\t\r \u00a0is\t\r \u00a0the\t\r \u00a0best\t\r \u00a0characterized.\t\r \u00a0It\t\r \u00a0is\t\r \u00a0located\t\r \u00a0at\t\r \u00a0centisome\t\r \u00a063\t\r \u00a0on\t\r \u00a0the\t\r \u00a0S.\t\r \u00a0Typhimurium\t\r \u00a0chromosome,\t\r \u00a0is\t\r \u00a040\t\r \u00a0kilo\t\r \u00a0base\t\r \u00a0pairs\t\r \u00a0(kb)\t\r \u00a0in\t\r \u00a0size,\t\r \u00a0and\t\r \u00a0encodes\t\r \u00a025\t\r \u00a0genes(23,\t\r \u00a024,\t\r \u00a088).\t\r \u00a0These\t\r \u00a0encode\t\r \u00a0for\t\r \u00a0regulatory\t\r \u00a0proteins,\t\r \u00a0proteins\t\r \u00a0that\t\r \u00a0form\t\r \u00a0a\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0system\t\r \u00a0(T3SS),\t\r \u00a0and\t\r \u00a0proteins\t\r \u00a0termed\t\r \u00a0translocons,\t\r \u00a0effectors\t\r \u00a0and\t\r \u00a0chaperones.\t\r \u00a0Strains\t\r \u00a0lacking\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0cause\t\r \u00a0delayed\t\r \u00a0gastroenteritis,\t\r \u00a0but\t\r \u00a0can\t\r \u00a0still\t\r \u00a0cause\t\r \u00a0enteric\t\r \u00a0fever(18,\t\r \u00a089-\u00ad\u201091).\t\r \u00a0It\t\r \u00a0is\t\r \u00a0present\t\r \u00a0in\t\r \u00a0all\t\r \u00a0Salmonella\t\r \u00a0species,\t\r \u00a0and\t\r \u00a0homologs\t\r \u00a0to\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0genes\t\r \u00a0exist\t\r \u00a0in\t\r \u00a0several\t\r \u00a0other\t\r \u00a0bacterial\t\r \u00a0pathogens.\t\r \u00a0Salmonella\t\r \u00a0pathogenicity\t\r \u00a0island\t\r \u00a02\t\r \u00a0(SPI-\u00ad\u20102)\t\r \u00a0is\t\r \u00a0located\t\r \u00a0at\t\r \u00a0centisome\t\r \u00a031,\t\r \u00a0is\t\r \u00a0 \t\r \u00a0\t\r \u00a0 11\t\r \u00a0 also\t\r \u00a040\t\r \u00a0kb\t\r \u00a0in\t\r \u00a0size,\t\r \u00a0and\t\r \u00a0contains\t\r \u00a0at\t\r \u00a0least\t\r \u00a017\t\r \u00a0genes(29,\t\r \u00a092,\t\r \u00a093).\t\r \u00a0Similar\t\r \u00a0to\t\r \u00a0SPI-\u00ad\u20101,\t\r \u00a0genes\t\r \u00a0within\t\r \u00a0SPI-\u00ad\u20102\t\r \u00a0encode\t\r \u00a0regulatory\t\r \u00a0proteins\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0components\t\r \u00a0of\t\r \u00a0a\t\r \u00a0T3SS,\t\r \u00a0translocons,\t\r \u00a0effectors,\t\r \u00a0and\t\r \u00a0chaperones.\t\r \u00a0SPI-\u00ad\u20102\t\r \u00a0is\t\r \u00a0required\t\r \u00a0for\t\r \u00a0systemic\t\r \u00a0illness,\t\r \u00a0and\t\r \u00a0it\t\r \u00a0is\t\r \u00a0only\t\r \u00a0present\t\r \u00a0in\t\r \u00a0Salmonella\t\r \u00a0enterica,\t\r \u00a0suggesting\t\r \u00a0that\t\r \u00a0it\t\r \u00a0was\t\r \u00a0acquired\t\r \u00a0later\t\r \u00a0in\t\r \u00a0evolution\t\r \u00a0than\t\r \u00a0SPI-\u00ad\u20101.\t\r \u00a0A\t\r \u00a0detailed\t\r \u00a0description\t\r \u00a0of\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0and\t\r \u00a0SPI-\u00ad\u20102\t\r \u00a0proteins\t\r \u00a0can\t\r \u00a0be\t\r \u00a0found\t\r \u00a0in\t\r \u00a0sections\t\r \u00a01.1.5\t\r \u00a0and\t\r \u00a01.1.6.\t\r \u00a0Numerous\t\r \u00a0smaller\t\r \u00a0pathogenicity\t\r \u00a0islets\t\r \u00a0also\t\r \u00a0confer\t\r \u00a0virulence\t\r \u00a0in\t\r \u00a0Salmonella\t\r \u00a0species.\t\r \u00a0Most\t\r \u00a0notable\t\r \u00a0of\t\r \u00a0these\t\r \u00a0is\t\r \u00a0the\t\r \u00a0SifA\t\r \u00a0gene,\t\r \u00a0which\t\r \u00a0is\t\r \u00a01.6\t\r \u00a0kb\t\r \u00a0in\t\r \u00a0size\t\r \u00a0and\t\r \u00a0located\t\r \u00a0at\t\r \u00a0centisome\t\r \u00a027(94).\t\r \u00a0SifA\t\r \u00a0is\t\r \u00a0unique\t\r \u00a0to\t\r \u00a0Salmonella\t\r \u00a0and\t\r \u00a0has\t\r \u00a0a\t\r \u00a0great\t\r \u00a0impact\t\r \u00a0on\t\r \u00a0virulence,\t\r \u00a0as\t\r \u00a0it\t\r \u00a0is\t\r \u00a0required\t\r \u00a0for\t\r \u00a0systemic\t\r \u00a0infection.\t\r \u00a0Specific\t\r \u00a0functions\t\r \u00a0and\t\r \u00a0regulation\t\r \u00a0of\t\r \u00a0SifA\t\r \u00a0are\t\r \u00a0described\t\r \u00a0in\t\r \u00a0relation\t\r \u00a0to\t\r \u00a0SPI-\u00ad\u20102\t\r \u00a0in\t\r \u00a0section\t\r \u00a01.1.6.4.\t\r \u00a0In\t\r \u00a0addition,\t\r \u00a0several\t\r \u00a0Salmonella\t\r \u00a0 enterica\t\r \u00a0serovars\t\r \u00a0also\t\r \u00a0contain\t\r \u00a0a\t\r \u00a0large\t\r \u00a0virulence\t\r \u00a0plasmid(95,\t\r \u00a096).\t\r \u00a0These\t\r \u00a0plasmids\t\r \u00a0differ\t\r \u00a0between\t\r \u00a0serovars,\t\r \u00a0but\t\r \u00a0all\t\r \u00a0contain\t\r \u00a0the\t\r \u00a08\t\r \u00a0kb\t\r \u00a0spv\t\r \u00a0operon\t\r \u00a0that\t\r \u00a0encodes\t\r \u00a0five\t\r \u00a0genes.\t\r \u00a0These\t\r \u00a0include\t\r \u00a0a\t\r \u00a0regulator\t\r \u00a0spvR,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0spvABCD.\t\r \u00a0It\t\r \u00a0was\t\r \u00a0originally\t\r \u00a0thought\t\r \u00a0that\t\r \u00a0virulence\t\r \u00a0plasmids\t\r \u00a0were\t\r \u00a0specific\t\r \u00a0to\t\r \u00a0non-\u00ad\u2010typhoidal\t\r \u00a0serovars.\t\r \u00a0However,\t\r \u00a0a\t\r \u00a0plasmid\t\r \u00a0containing\t\r \u00a0spv\t\r \u00a0genes\t\r \u00a0has\t\r \u00a0now\t\r \u00a0been\t\r \u00a0identified\t\r \u00a0in\t\r \u00a0S.\t\r \u00a0Typhi\t\r \u00a0as\t\r \u00a0well(97).\t\r \u00a0Specific\t\r \u00a0functions\t\r \u00a0of\t\r \u00a0spvB\t\r \u00a0will\t\r \u00a0be\t\r \u00a0described\t\r \u00a0along\t\r \u00a0with\t\r \u00a0SPI-\u00ad\u20102\t\r \u00a0in\t\r \u00a0section\t\r \u00a01.1.6.4.\t\r \u00a0Genes\t\r \u00a0within\t\r \u00a0pathogenicity\t\r \u00a0islands\t\r \u00a0and\t\r \u00a0islets\t\r \u00a0must\t\r \u00a0be\t\r \u00a0regulated\t\r \u00a0and\t\r \u00a0expressed\t\r \u00a0at\t\r \u00a0specific\t\r \u00a0stages\t\r \u00a0during\t\r \u00a0infection\t\r \u00a0to\t\r \u00a0confer\t\r \u00a0virulence.\t\r \u00a0Several\t\r \u00a0of\t\r \u00a0these\t\r \u00a0gene\t\r \u00a0clusters\t\r \u00a0contain\t\r \u00a0transcriptional\t\r \u00a0regulators\t\r \u00a0that\t\r \u00a0were\t\r \u00a0acquired\t\r \u00a0together\t\r \u00a0with\t\r \u00a0virulence\t\r \u00a0genes.\t\r \u00a0For\t\r \u00a0example,\t\r \u00a0HilA\t\r \u00a0and\t\r \u00a0InvF\t\r \u00a0are\t\r \u00a0within\t\r \u00a0SPI-\u00ad\u20101,\t\r \u00a0SsrA\t\r \u00a0and\t\r \u00a0SsrB\t\r \u00a0are\t\r \u00a0within\t\r \u00a0the\t\r \u00a0SPI-\u00ad\u20102\t\r \u00a0island,\t\r \u00a0and\t\r \u00a0SpvR\t\r \u00a0is\t\r \u00a0located\t\r \u00a0within\t\r \u00a0the\t\r \u00a0spv\t\r \u00a0operon\t\r \u00a0in\t\r \u00a0Salmonella\t\r \u00a0virulence\t\r \u00a0plasmids.\t\r \u00a0However,\t\r \u00a0the\t\r \u00a0islands\t\r \u00a0and\t\r \u00a0islets\t\r \u00a0themselves\t\r \u00a0are\t\r \u00a0often\t\r \u00a0regulated\t\r \u00a0 \t\r \u00a0\t\r \u00a0 12\t\r \u00a0 by\t\r \u00a0systems\t\r \u00a0that\t\r \u00a0were\t\r \u00a0present\t\r \u00a0in\t\r \u00a0the\t\r \u00a0bacterial\t\r \u00a0genome\t\r \u00a0before\t\r \u00a0the\t\r \u00a0acquisition\t\r \u00a0of\t\r \u00a0the\t\r \u00a0virulence\t\r \u00a0genes.\t\r \u00a0The\t\r \u00a0PhoP\/PhoQ\t\r \u00a0two-\u00ad\u2010component\t\r \u00a0regulatory\t\r \u00a0system\t\r \u00a0is\t\r \u00a0one\t\r \u00a0example,\t\r \u00a0and\t\r \u00a0has\t\r \u00a0profound\t\r \u00a0impact\t\r \u00a0Salmonella\t\r \u00a0virulence.\t\r \u00a0PhoP\t\r \u00a0and\t\r \u00a0PhoQ\t\r \u00a0exist\t\r \u00a0in\t\r \u00a0several\t\r \u00a0pathogenic\t\r \u00a0and\t\r \u00a0non-\u00ad\u2010pathogenic\t\r \u00a0bacterial\t\r \u00a0species.\t\r \u00a0They\t\r \u00a0encode\t\r \u00a0a\t\r \u00a0sensor\t\r \u00a0histidine\t\r \u00a0kinase\t\r \u00a0(PhoQ)\t\r \u00a0which\t\r \u00a0is\t\r \u00a0autophosphorylated\t\r \u00a0under\t\r \u00a0specific\t\r \u00a0environmental\t\r \u00a0conditions(98).\t\r \u00a0The\t\r \u00a0phosphate\t\r \u00a0is\t\r \u00a0then\t\r \u00a0transferred\t\r \u00a0to\t\r \u00a0an\t\r \u00a0aspartic\t\r \u00a0acid\t\r \u00a0residue\t\r \u00a0in\t\r \u00a0PhoP,\t\r \u00a0which\t\r \u00a0activates\t\r \u00a0it\t\r \u00a0as\t\r \u00a0a\t\r \u00a0transcriptional\t\r \u00a0regulator.\t\r \u00a0In\t\r \u00a0Salmonella,\t\r \u00a0PhoQ\t\r \u00a0is\t\r \u00a0inactivated\t\r \u00a0at\t\r \u00a0high\t\r \u00a0concentrations\t\r \u00a0of\t\r \u00a0divalent\t\r \u00a0cations,\t\r \u00a0and\t\r \u00a0activated\t\r \u00a0at\t\r \u00a0low\t\r \u00a0pH\t\r \u00a0and\t\r \u00a0by\t\r \u00a0cationic\t\r \u00a0antimicrobial\t\r \u00a0peptides(98).\t\r \u00a0PhoP\t\r \u00a0regulates\t\r \u00a0expression\t\r \u00a0of\t\r \u00a0over\t\r \u00a040\t\r \u00a0genes.\t\r \u00a0These\t\r \u00a0include\t\r \u00a0repression\t\r \u00a0of\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0genes,\t\r \u00a0and\t\r \u00a0activation\t\r \u00a0of\t\r \u00a0genes\t\r \u00a0for\t\r \u00a0defense\t\r \u00a0against\t\r \u00a0host\t\r \u00a0antimicrobials(99,\t\r \u00a0100).\t\r \u00a0While\t\r \u00a0its\t\r \u00a0role\t\r \u00a0in\t\r \u00a0regulating\t\r \u00a0SPI-\u00ad\u20102\t\r \u00a0remains\t\r \u00a0controversial,\t\r \u00a0stains\t\r \u00a0lacking\t\r \u00a0PhoP\t\r \u00a0are\t\r \u00a0severely\t\r \u00a0attenuated\t\r \u00a0for\t\r \u00a0systemic\t\r \u00a0infection(101-\u00ad\u2010103).\t\r \u00a0\t\r \u00a0 1.1.5\t\r \u00a0Salmonella-\u00ad\u2010pathogenicity\t\r \u00a0island\t\r \u00a01\t\r \u00a0 1.1.5.1\t\r \u00a0Regulation\t\r \u00a0of\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0is\t\r \u00a0essential\t\r \u00a0to\t\r \u00a0all\t\r \u00a0forms\t\r \u00a0of\t\r \u00a0Salmonellosis\t\r \u00a0and\t\r \u00a0is\t\r \u00a0present\t\r \u00a0in\t\r \u00a0all\t\r \u00a0Salmonella\t\r \u00a0species(104,\t\r \u00a0105).\t\r \u00a0Genes\t\r \u00a0within\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0are\t\r \u00a0primarily\t\r \u00a0expressed\t\r \u00a0within\t\r \u00a0the\t\r \u00a0host\t\r \u00a0intestinal\t\r \u00a0lumen\t\r \u00a0and\t\r \u00a0epithelium\t\r \u00a0to\t\r \u00a0facilitate\t\r \u00a0invasion\t\r \u00a0of\t\r \u00a0non-\u00ad\u2010phagocytic\t\r \u00a0cells(106,\t\r \u00a0107).\t\r \u00a0Many\t\r \u00a0environmental\t\r \u00a0factors\t\r \u00a0are\t\r \u00a0thought\t\r \u00a0to\t\r \u00a0regulate\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0gene\t\r \u00a0expression,\t\r \u00a0but\t\r \u00a0the\t\r \u00a0specific\t\r \u00a0mechanisms\t\r \u00a0involved\t\r \u00a0are\t\r \u00a0not\t\r \u00a0well\t\r \u00a0understood.\t\r \u00a0Changes\t\r \u00a0induced\t\r \u00a0when\t\r \u00a0bacteria\t\r \u00a0grow\t\r \u00a0to\t\r \u00a0high\t\r \u00a0density,\t\r \u00a0and\t\r \u00a0conditions\t\r \u00a0such\t\r \u00a0as\t\r \u00a0high\t\r \u00a0osmolarity,\t\r \u00a0low\t\r \u00a0aeration,\t\r \u00a0and\t\r \u00a0slightly\t\r \u00a0basic\t\r \u00a0pH\t\r \u00a0have\t\r \u00a0been\t\r \u00a0suggested\t\r \u00a0for\t\r \u00a0optimal\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0gene\t\r \u00a0 \t\r \u00a0\t\r \u00a0 13\t\r \u00a0 expression(108-\u00ad\u2010111).\t\r \u00a0In\t\r \u00a0parallel,\t\r \u00a0regulatory\t\r \u00a0systems\t\r \u00a0such\t\r \u00a0as\t\r \u00a0EnvZ\/OmpR\t\r \u00a0and\t\r \u00a0Bar\/SirA\t\r \u00a0have\t\r \u00a0also\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0up-\u00ad\u2010regulate\t\r \u00a0SPI-\u00ad\u20101,\t\r \u00a0but\t\r \u00a0connections\t\r \u00a0between\t\r \u00a0these\t\r \u00a0and\t\r \u00a0environmental\t\r \u00a0factors\t\r \u00a0have\t\r \u00a0not\t\r \u00a0been\t\r \u00a0well\t\r \u00a0established(112,\t\r \u00a0113).\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0contains\t\r \u00a0all\t\r \u00a0the\t\r \u00a0genes\t\r \u00a0necessary\t\r \u00a0to\t\r \u00a0create\t\r \u00a0a\t\r \u00a0functional\t\r \u00a0T3SS\t\r \u00a0apparatus,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0several\t\r \u00a0effectors\t\r \u00a0and\t\r \u00a0regulators.\t\r \u00a0These\t\r \u00a0are\t\r \u00a0encoded\t\r \u00a0by\t\r \u00a0three\t\r \u00a0operons,\t\r \u00a0the\t\r \u00a0prg\/org\t\r \u00a0operon,\t\r \u00a0the\t\r \u00a0inv\/spa\t\r \u00a0operon,\t\r \u00a0and\t\r \u00a0the\t\r \u00a0sic\/sip\t\r \u00a0operon.\t\r \u00a0The\t\r \u00a0prg\/org\t\r \u00a0and\t\r \u00a0inv\/spa\t\r \u00a0operons\t\r \u00a0encode\t\r \u00a0components\t\r \u00a0of\t\r \u00a0the\t\r \u00a0T3SS\t\r \u00a0needle\t\r \u00a0complex,\t\r \u00a0and\t\r \u00a0the\t\r \u00a0sip\/sic\t\r \u00a0operon\t\r \u00a0encodes\t\r \u00a0proteins\t\r \u00a0required\t\r \u00a0for\t\r \u00a0effector\t\r \u00a0translocation\t\r \u00a0into\t\r \u00a0host\t\r \u00a0cells(109,\t\r \u00a0114).\t\r \u00a0Several\t\r \u00a0transcriptional\t\r \u00a0regulators\t\r \u00a0govern\t\r \u00a0expression\t\r \u00a0of\t\r \u00a0these\t\r \u00a0genes,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0several\t\r \u00a0outside\t\r \u00a0of\t\r \u00a0SPI-\u00ad\u20101.\t\r \u00a0Initially,\t\r \u00a0HilD\t\r \u00a0is\t\r \u00a0thought\t\r \u00a0to\t\r \u00a0mediate\t\r \u00a0most\t\r \u00a0external\t\r 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\u00a0as\t\r \u00a0PhoP\/PhoQ,\t\r \u00a0PhoR\/PhoB,\t\r \u00a0and\t\r \u00a0FimZ\/FimY\t\r \u00a0that\t\r \u00a0negatively\t\r \u00a0regulate\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0are\t\r \u00a0thought\t\r \u00a0to\t\r \u00a0target\t\r \u00a0HilE(115-\u00ad\u2010117).\t\r \u00a0\t\r \u00a0 1.1.5.2\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0apparatus\t\r \u00a0T3SSs\t\r \u00a0are\t\r \u00a0specialized\t\r \u00a0protein\t\r \u00a0systems\t\r \u00a0developed\t\r \u00a0to\t\r \u00a0secrete\t\r \u00a0bacterial\t\r \u00a0effector\t\r \u00a0proteins\t\r \u00a0into\t\r \u00a0host\t\r \u00a0cells.\t\r \u00a0These\t\r \u00a0systems\t\r \u00a0are\t\r \u00a0composed\t\r \u00a0of\t\r \u00a0over\t\r \u00a020\t\r \u00a0proteins,\t\r \u00a0which\t\r \u00a0are\t\r \u00a0conserved\t\r \u00a0among\t\r \u00a0animal\t\r \u00a0and\t\r \u00a0plant\t\r \u00a0pathogens,\t\r \u00a0and\t\r \u00a0also\t\r \u00a0evolutionarily\t\r \u00a0related\t\r \u00a0to\t\r \u00a0components\t\r \u00a0of\t\r \u00a0the\t\r 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\u00a0been\t\r \u00a0done\t\r \u00a0to\t\r \u00a0define\t\r \u00a0the\t\r \u00a0organization\t\r \u00a0and\t\r \u00a0specific\t\r \u00a0components\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0T3SS\t\r \u00a0apparatus(121-\u00ad\u2010127)).\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0contains\t\r \u00a0two\t\r \u00a0distinct\t\r \u00a0groups\t\r \u00a0of\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0associated\t\r \u00a0proteins(118,\t\r \u00a0119).\t\r \u00a0One\t\r \u00a0group\t\r \u00a0contains\t\r \u00a0inner\t\r \u00a0membrane\t\r \u00a0proteins\t\r \u00a0that\t\r \u00a0form\t\r \u00a0the\t\r \u00a0export\t\r \u00a0machinery.\t\r \u00a0These\t\r \u00a0proteins\t\r \u00a0have\t\r \u00a0close\t\r \u00a0homologs\t\r \u00a0in\t\r \u00a0the\t\r \u00a0flagellar\t\r \u00a0system,\t\r \u00a0and\t\r \u00a0are\t\r \u00a0thought\t\r \u00a0to\t\r \u00a0facilitate\t\r \u00a0engagement\t\r \u00a0and\t\r \u00a0transport\t\r \u00a0through\t\r \u00a0the\t\r \u00a0apparatus.\t\r \u00a0A\t\r \u00a0second\t\r \u00a0group\t\r \u00a0has\t\r \u00a0very\t\r \u00a0little\t\r \u00a0similarity\t\r \u00a0to\t\r \u00a0components\t\r \u00a0of\t\r \u00a0the\t\r \u00a0flagellar\t\r \u00a0system,\t\r \u00a0and\t\r \u00a0form\t\r \u00a0the\t\r \u00a0syringe\t\r \u00a0like\t\r \u00a0needle\t\r \u00a0complex.\t\r \u00a0\t\r \u00a0Assembly\t\r \u00a0of\t\r \u00a0the\t\r \u00a0system\t\r \u00a0is\t\r \u00a0thought\t\r \u00a0to\t\r \u00a0occur\t\r \u00a0in\t\r \u00a0three\t\r \u00a0phases(123,\t\r \u00a0128).\t\r \u00a0During\t\r \u00a0the\t\r \u00a0first\t\r \u00a0phase,\t\r \u00a0components\t\r \u00a0forming\t\r \u00a0the\t\r \u00a0base\t\r \u00a0of\t\r \u00a0the\t\r \u00a0needle\t\r \u00a0complex\t\r \u00a0are\t\r \u00a0exported\t\r \u00a0through\t\r \u00a0the\t\r \u00a0inner\t\r \u00a0bacterial\t\r \u00a0membrane\t\r \u00a0by\t\r \u00a0Sec-\u00ad\u2010dependent\t\r \u00a0transport.\t\r \u00a0Initially\t\r \u00a0PrgH\t\r \u00a0and\t\r \u00a0PrgK\t\r \u00a0form\t\r \u00a0two\t\r \u00a0hollow\t\r \u00a0rings,\t\r \u00a0which\t\r \u00a0localize\t\r \u00a0within\t\r \u00a0the\t\r \u00a0inner\t\r \u00a0bacterial\t\r \u00a0membrane.\t\r \u00a0Subsequently,\t\r \u00a0InvG\t\r \u00a0is\t\r \u00a0also\t\r \u00a0secreted\t\r \u00a0by\t\r \u00a0Sec-\u00ad\u2010dependent\t\r \u00a0transport\t\r \u00a0to\t\r \u00a0form\t\r \u00a0rings\t\r \u00a0within\t\r \u00a0the\t\r \u00a0outer\t\r \u00a0membrane\t\r \u00a0by\t\r \u00a0a\t\r \u00a0process\t\r \u00a0that\t\r \u00a0is\t\r \u00a0aided\t\r \u00a0by\t\r \u00a0InvH.\t\r \u00a0Together,\t\r \u00a0PrgH,\t\r \u00a0PrgK\t\r \u00a0and\t\r \u00a0InvG\t\r \u00a0form\t\r \u00a0the\t\r \u00a0base\t\r \u00a0of\t\r \u00a0the\t\r \u00a0needle\t\r \u00a0complex.\t\r \u00a0The\t\r \u00a0second\t\r \u00a0phase\t\r \u00a0of\t\r \u00a0assembly\t\r \u00a0 \t\r \u00a0\t\r \u00a0 15\t\r \u00a0 involves\t\r \u00a0formation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0type\t\r \u00a0III\t\r \u00a0export\t\r \u00a0apparatus,\t\r \u00a0which\t\r \u00a0contains\t\r \u00a0at\t\r \u00a0least\t\r \u00a0eight\t\r \u00a0proteins\t\r \u00a0(SpaO,\t\r \u00a0P,\t\r \u00a0Q,\t\r \u00a0R,\t\r \u00a0S\t\r \u00a0and\t\r \u00a0InvA,\t\r \u00a0InvC,\t\r \u00a0and\t\r \u00a0OrgA),\t\r \u00a0one\t\r \u00a0of\t\r \u00a0which\t\r \u00a0(InvC)\t\r \u00a0is\t\r \u00a0an\t\r \u00a0ATPase,\t\r \u00a0and\t\r \u00a0several\t\r \u00a0of\t\r \u00a0which\t\r \u00a0are\t\r \u00a0membrane\t\r \u00a0proteins\t\r \u00a0predicted\t\r \u00a0to\t\r \u00a0sit\t\r \u00a0within\t\r \u00a0the\t\r \u00a0hollow\t\r \u00a0of\t\r \u00a0the\t\r \u00a0base(129).\t\r \u00a0The\t\r \u00a0third\t\r \u00a0phase\t\r \u00a0of\t\r \u00a0assembly\t\r \u00a0involves\t\r \u00a0sequential\t\r \u00a0(export\t\r \u00a0apparatus\t\r \u00a0dependent)\t\r \u00a0secretion\t\r \u00a0of\t\r \u00a0components\t\r \u00a0through\t\r \u00a0the\t\r \u00a0center\t\r \u00a0of\t\r \u00a0the\t\r \u00a0complex\t\r \u00a0and\t\r \u00a0their\t\r \u00a0assembly\t\r \u00a0at\t\r \u00a0the\t\r \u00a0growing\t\r \u00a0edge.\t\r \u00a0Four\t\r \u00a0proteins\t\r \u00a0are\t\r \u00a0required\t\r \u00a0for\t\r \u00a0formation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0needle,\t\r \u00a0which\t\r \u00a0extends\t\r \u00a0out\t\r \u00a0beyond\t\r \u00a0the\t\r \u00a0outer\t\r \u00a0membrane.\t\r \u00a0These\t\r \u00a0include\t\r \u00a0PrgI,\t\r \u00a0which\t\r \u00a0is\t\r \u00a0the\t\r \u00a0main\t\r \u00a0component\t\r \u00a0of\t\r \u00a0the\t\r \u00a0needle\t\r \u00a0structure,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0PrgJ,\t\r \u00a0which\t\r \u00a0regulates\t\r \u00a0needle\t\r \u00a0length,\t\r \u00a0and\t\r \u00a0InvI\t\r \u00a0and\t\r \u00a0OrgA\t\r \u00a0with\t\r \u00a0currently\t\r \u00a0unknown\t\r \u00a0function.\t\r \u00a0The\t\r \u00a0fully\t\r \u00a0formed\t\r \u00a0needle\t\r \u00a0complex\t\r \u00a0is\t\r \u00a0approximately\t\r \u00a020\t\r \u00a0nm\t\r \u00a0in\t\r \u00a0width\t\r \u00a0and\t\r \u00a080\t\r \u00a0nm\t\r \u00a0in\t\r \u00a0height.\t\r \u00a0\t\r \u00a0 1.1.5.3\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0translocons\t\r \u00a0Once\t\r \u00a0the\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0needle\t\r \u00a0complex\t\r \u00a0is\t\r \u00a0assembled,\t\r \u00a0several\t\r \u00a0effector\t\r \u00a0proteins\t\r \u00a0are\t\r \u00a0transported\t\r \u00a0through\t\r \u00a0the\t\r \u00a0apparatus\t\r \u00a0and\t\r \u00a0into\t\r \u00a0host\t\r \u00a0cells.\t\r \u00a0This\t\r \u00a0requires\t\r \u00a0three\t\r \u00a0proteins\t\r \u00a0termed\t\r \u00a0translocons,\t\r \u00a0which\t\r \u00a0are\t\r \u00a0secreted\t\r \u00a0by\t\r \u00a0the\t\r \u00a0apparatus\t\r \u00a0and\t\r \u00a0inserted\t\r \u00a0into\t\r \u00a0the\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0membrane(130).\t\r \u00a0These\t\r \u00a0proteins\t\r \u00a0(SipB,\t\r \u00a0SipC\t\r \u00a0and\t\r \u00a0SipD)\t\r \u00a0allow\t\r \u00a0intimate\t\r \u00a0contact\t\r \u00a0with\t\r \u00a0host\t\r \u00a0cells,\t\r \u00a0and\t\r \u00a0are\t\r \u00a0required\t\r \u00a0for\t\r \u00a0subsequent\t\r \u00a0transport\t\r \u00a0of\t\r \u00a0effectors\t\r \u00a0and\t\r \u00a0chaperones\t\r \u00a0into\t\r \u00a0the\t\r \u00a0host\t\r \u00a0cytosol(131-\u00ad\u2010135).\t\r \u00a0Little\t\r \u00a0is\t\r \u00a0known\t\r \u00a0about\t\r \u00a0the\t\r \u00a0structure\t\r \u00a0of\t\r \u00a0the\t\r \u00a0translocon\t\r \u00a0apparatus.\t\r \u00a0However,\t\r \u00a0it\t\r \u00a0has\t\r \u00a0been\t\r \u00a0proposed\t\r \u00a0that\t\r \u00a0these\t\r \u00a0proteins\t\r \u00a0form\t\r \u00a0an\t\r \u00a0oligomeric\t\r \u00a0protein\t\r \u00a0channel\t\r \u00a0that\t\r \u00a0inserts\t\r \u00a0into\t\r \u00a0the\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0membrane.\t\r \u00a0This\t\r \u00a0is\t\r \u00a0supported\t\r \u00a0by\t\r \u00a0the\t\r \u00a0fact\t\r \u00a0that\t\r \u00a0deletion\t\r \u00a0of\t\r \u00a0any\t\r \u00a0of\t\r \u00a0the\t\r \u00a0three\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0translocon\t\r \u00a0proteins\t\r \u00a0inhibits\t\r \u00a0translocation(136).\t\r \u00a0Furthermore,\t\r \u00a0SipC\t\r \u00a0cannot\t\r \u00a0be\t\r \u00a0detected\t\r \u00a0on\t\r \u00a0the\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0membrane\t\r \u00a0in\t\r \u00a0the\t\r \u00a0absence\t\r \u00a0of\t\r \u00a0SipB,\t\r \u00a0suggesting\t\r \u00a0that\t\r \u00a0pre-\u00ad\u2010assembly\t\r \u00a0of\t\r \u00a0a\t\r \u00a0complex\t\r \u00a0is\t\r \u00a0 \t\r \u00a0\t\r \u00a0 16\t\r \u00a0 required\t\r \u00a0for\t\r \u00a0membrane\t\r \u00a0insertion(137).\t\r \u00a0In\t\r \u00a0addition,\t\r \u00a0cholesterol\t\r \u00a0levels\t\r \u00a0are\t\r \u00a0also\t\r \u00a0thought\t\r \u00a0to\t\r \u00a0impact\t\r \u00a0translocon\t\r \u00a0formation\t\r \u00a0and\t\r \u00a0function,\t\r \u00a0as\t\r \u00a0SipB\t\r \u00a0binds\t\r \u00a0cholesterol\t\r \u00a0directly\t\r \u00a0and\t\r \u00a0low\t\r \u00a0levels\t\r \u00a0of\t\r \u00a0cholesterol\t\r \u00a0reduce\t\r \u00a0the\t\r \u00a0amount\t\r \u00a0of\t\r \u00a0translocons\t\r \u00a0on\t\r \u00a0the\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0membrane(138).\t\r \u00a0\t\r \u00a0 1.1.5.4\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0effectors\t\r \u00a0Eleven\t\r \u00a0effector\t\r \u00a0proteins\t\r \u00a0are\t\r \u00a0known\t\r \u00a0to\t\r \u00a0be\t\r \u00a0secreted\t\r \u00a0by\t\r \u00a0Salmonella\t\r \u00a0T3SS-\u00ad\u20101(21).\t\r \u00a0These\t\r \u00a0proteins\t\r \u00a0are\t\r \u00a0translocated\t\r \u00a0into\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0where\t\r \u00a0they\t\r \u00a0mimic\t\r \u00a0host\t\r \u00a0protein\t\r \u00a0functions\t\r \u00a0to\t\r \u00a0induce\t\r \u00a0an\t\r \u00a0array\t\r \u00a0of\t\r \u00a0cellular\t\r \u00a0processes\t\r \u00a0(Table\t\r \u00a01.1)(139).\t\r \u00a0Bacterial\t\r \u00a0domains\t\r \u00a0exerting\t\r \u00a0these\t\r \u00a0activities\t\r \u00a0are\t\r \u00a0rarely\t\r \u00a0homologous\t\r \u00a0to\t\r \u00a0those\t\r \u00a0found\t\r \u00a0in\t\r \u00a0eukaryotes,\t\r \u00a0suggesting\t\r \u00a0that\t\r \u00a0have\t\r \u00a0evolved\t\r \u00a0independently.\t\r \u00a0Most\t\r \u00a0of\t\r \u00a0these\t\r \u00a0effectors\t\r \u00a0are\t\r \u00a0specific\t\r \u00a0to\t\r \u00a0secretion\t\r \u00a0by\t\r \u00a0T3SS-\u00ad\u20101.\t\r \u00a0However,\t\r \u00a0two\t\r \u00a0(SspH1\t\r \u00a0and\t\r \u00a0SlrP)\t\r \u00a0are\t\r \u00a0also\t\r \u00a0secreted\t\r \u00a0by\t\r \u00a0the\t\r \u00a0SPI-\u00ad\u20102\t\r \u00a0encoded\t\r \u00a0secretion\t\r \u00a0system(140).\t\r \u00a0\t\r \u00a0 Table\t\r \u00a01.1\t\r \u00a0Functions\t\r \u00a0and\t\r \u00a0host\t\r \u00a0targets\t\r \u00a0of\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0effectors\t\r \u00a0 Effector\t\r \u00a0 Cellular\t\r \u00a0activity\t\r \u00a0 Host-\u00ad\u2010target\t\r \u00a0 Reference\t\r \u00a0AvrA\t\r \u00a0 Inhibits\t\r \u00a0NF-\u00ad\u2010\u03baB\t\r \u00a0through\t\r \u00a0deubiquitylation\t\r \u00a0of\t\r \u00a0I-\u00ad\u2010 \u03baB\u03b1\t\r \u00a0and\t\r \u00a0\u03b2-\u00ad\u2010catenin.\t\r \u00a0Inhibits\t\r \u00a0JNK\t\r \u00a0through\t\r \u00a0acetylation\t\r \u00a0of\t\r \u00a0MEK7.\t\r \u00a0 MEK7,\t\r \u00a0I-\u00ad\u2010\u03baB\u03b1,\t\r \u00a0\u03b2-\u00ad\u2010catenin\t\r \u00a0 (141,\t\r \u00a0142)\t\r \u00a0(143,\t\r \u00a0144)\t\r \u00a0 SipA\/SspA\t\r \u00a0 Promotes\t\r \u00a0formation\t\r \u00a0of\t\r \u00a0F-\u00ad\u2010actin,\t\r \u00a0tight-\u00ad\u2010junction\t\r \u00a0disruption,\t\r \u00a0PMN\t\r \u00a0migration,\t\r \u00a0PKC\t\r \u00a0and\t\r \u00a0caspase-\u00ad\u20103\t\r \u00a0activation,\t\r \u00a0and\t\r \u00a0SCV\t\r \u00a0maturation.\t\r \u00a0 actin,\t\r \u00a0T-\u00ad\u2010plastin\t\r \u00a0 (145-\u00ad\u2010150)\t\r \u00a0 SipB\/SspB\t\r \u00a0 Binds\t\r \u00a0to\t\r \u00a0caspase-\u00ad\u20101\t\r \u00a0and\t\r \u00a0induces\t\r \u00a0autophagy\t\r \u00a0and\t\r \u00a0cell\t\r \u00a0death\t\r \u00a0in\t\r \u00a0macrophages.\t\r \u00a0 caspase-\u00ad\u20101,\t\r \u00a0cholesterol\t\r \u00a0 (39,\t\r \u00a0138,\t\r \u00a0151-\u00ad\u2010153)\t\r \u00a0 SipC\/SspC\t\r \u00a0 Bundles\t\r \u00a0F-\u00ad\u2010actin\t\r \u00a0and\t\r \u00a0nucleates\t\r \u00a0actin\t\r \u00a0polymerization.\t\r \u00a0 actin,\t\r \u00a0cytokeratin-\u00ad\u201018\t\r \u00a0 (154,\t\r \u00a0155)\t\r \u00a0(156-\u00ad\u2010159)\t\r \u00a0 \t\r \u00a0\t\r \u00a0 17\t\r \u00a0 Effector\t\r \u00a0 Cellular\t\r \u00a0activity\t\r \u00a0 Host-\u00ad\u2010target\t\r \u00a0 Reference\t\r \u00a0SopA\t\r \u00a0 Contributes\t\r \u00a0to\t\r \u00a0invasion,\t\r \u00a0localizes\t\r \u00a0to\t\r \u00a0mitochondria,\t\r \u00a0and\t\r \u00a0stimulates\t\r \u00a0transepithelial\t\r \u00a0migration\t\r \u00a0through\t\r \u00a0E3\t\r \u00a0ubiquitin\t\r \u00a0ligase\t\r \u00a0activity.\t\r \u00a0 unknown\t\r \u00a0 (160-\u00ad\u2010162)\t\r \u00a0 SopB\/SigD\t\r \u00a0 Activates\t\r \u00a0Cdc42,\t\r \u00a0RhoG\t\r \u00a0and\t\r \u00a0Akt\t\r \u00a0(phosphoinositide\t\r \u00a0phosphatase).\t\r \u00a0Regulates\t\r \u00a0formation\t\r \u00a0and\t\r \u00a0trafficking\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV,\t\r \u00a0chloride\t\r \u00a0secretion,\t\r \u00a0disruption\t\r \u00a0of\t\r \u00a0tight\t\r \u00a0junctions,\t\r \u00a0and\t\r \u00a0anti-\u00ad\u2010apoptosis.\t\r \u00a0 Cdc42\t\r \u00a0 (45,\t\r \u00a057,\t\r \u00a058,\t\r \u00a066,\t\r \u00a0148,\t\r \u00a0163-\u00ad\u2010166)\t\r \u00a0 SopD\t\r \u00a0 Contributes\t\r \u00a0to\t\r \u00a0invasion,\t\r \u00a0cooperates\t\r \u00a0with\t\r \u00a0SopB\t\r \u00a0for\t\r \u00a0macropinosome\t\r \u00a0formation\t\r \u00a0and\t\r \u00a0membrane\t\r \u00a0fission.\t\r \u00a0 unknown\t\r \u00a0 (160,\t\r \u00a0167)\t\r \u00a0 SopE\t\r \u00a0 Acts\t\r \u00a0as\t\r \u00a0a\t\r \u00a0GEF\t\r \u00a0to\t\r \u00a0activate\t\r \u00a0Cdc42,\t\r \u00a0Rac1,\t\r \u00a0RhoG\t\r \u00a0and\t\r \u00a0Rab5.\t\r \u00a0Activates\t\r \u00a0caspase-\u00ad\u20101\t\r \u00a0and\t\r \u00a0disrupts\t\r \u00a0tight\t\r \u00a0junctions.\t\r \u00a0 Cdc42,\t\r \u00a0Rac1,\t\r \u00a0RhoG\t\r \u00a0 (45,\t\r \u00a0148,\t\r \u00a0168-\u00ad\u2010172)\t\r \u00a0 SopE2\t\r \u00a0 Activates\t\r \u00a0Cdc42\t\r \u00a0through\t\r \u00a0its\t\r \u00a0GEF\t\r \u00a0activity.\t\r \u00a0Disrupts\t\r \u00a0tight\t\r \u00a0junctions.\t\r \u00a0 Cdc42\t\r \u00a0 (148,\t\r \u00a0173,\t\r \u00a0174)\t\r \u00a0SptP\t\r \u00a0 Inhibits\t\r \u00a0Rac1\t\r \u00a0and\t\r \u00a0Cdc42\t\r \u00a0(GAP),\t\r \u00a0and\t\r \u00a0MAPK\t\r \u00a0signaling\t\r \u00a0(PTP).\t\r \u00a0Dephosphorylates\t\r \u00a0VCP\t\r \u00a0to\t\r \u00a0promote\t\r \u00a0bacterial\t\r \u00a0replication.\t\r \u00a0 Cdc42,\t\r \u00a0Rac1,\t\r \u00a0Raf1,\t\r \u00a0VCP\t\r \u00a0 (175-\u00ad\u2010179)\t\r \u00a0 SlrP\t\r \u00a0 E3\t\r \u00a0ubiquitin\t\r \u00a0ligase.\t\r \u00a0Targets\t\r \u00a0thioredoxin\t\r \u00a0for\t\r \u00a0degradation,\t\r \u00a0promoting\t\r \u00a0cell\t\r \u00a0death.\t\r \u00a0Also\t\r \u00a0targets\t\r \u00a0ER\t\r \u00a0chaperone\t\r \u00a0ERdj3.\t\r \u00a0 thioredoxin,\t\r \u00a0ERdj3\t\r \u00a0 (180,\t\r \u00a0181)\t\r \u00a0 SspH1\t\r \u00a0 Inhibits\t\r \u00a0NF-\u00ad\u2010\u03baB\t\r \u00a0dependent\t\r \u00a0gene\t\r \u00a0expression.\t\r \u00a0Interacts\t\r \u00a0with\t\r \u00a0PKN1\t\r \u00a0and\t\r \u00a0had\t\r \u00a0E3\t\r \u00a0ubiquitin\t\r \u00a0ligase\t\r \u00a0activity.\t\r \u00a0 PKN1\t\r \u00a0 (182-\u00ad\u2010184)\t\r \u00a0 \t\r \u00a0 1.1.5.4.1\t\r \u00a0Salmonella-\u00ad\u2010induced\t\r \u00a0endocytosis\t\r \u00a0The\t\r \u00a0most\t\r \u00a0extensively\t\r \u00a0studied\t\r \u00a0action\t\r \u00a0of\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0effectors\t\r \u00a0is\t\r \u00a0their\t\r \u00a0ability\t\r \u00a0to\t\r \u00a0induce\t\r \u00a0extensive\t\r \u00a0actin\t\r \u00a0rearrangements\t\r \u00a0leading\t\r \u00a0to\t\r \u00a0membrane\t\r \u00a0ruffling\t\r \u00a0and\t\r \u00a0 \t\r \u00a0\t\r \u00a0 18\t\r \u00a0 macropinocytosis\t\r \u00a0in\t\r \u00a0non-\u00ad\u2010phagocytic\t\r \u00a0cells.\t\r \u00a0This\t\r \u00a0is\t\r \u00a0dependent\t\r \u00a0upon\t\r \u00a0the\t\r \u00a0effector\t\r \u00a0proteins\t\r \u00a0SopB\t\r \u00a0(also\t\r \u00a0known\t\r \u00a0as\t\r \u00a0SigD)\t\r \u00a0and\t\r \u00a0SopE\/E2,\t\r \u00a0which\t\r \u00a0activate\t\r \u00a0small\t\r \u00a0GTPases\t\r \u00a0Rac1\t\r \u00a0and\t\r \u00a0RhoG.\t\r \u00a0SopE\/E2\t\r \u00a0are\t\r \u00a0guanine\t\r \u00a0nucleotide\t\r \u00a0exchange\t\r \u00a0factors\t\r \u00a0(GEFs),\t\r \u00a0and\t\r \u00a0thus\t\r \u00a0activate\t\r \u00a0these\t\r \u00a0small\t\r \u00a0GTPases\t\r \u00a0directly,\t\r \u00a0while\t\r \u00a0SopB\t\r \u00a0is\t\r \u00a0a\t\r \u00a0phosphoinositide\t\r \u00a0phosphatase\t\r \u00a0and\t\r \u00a0is\t\r \u00a0thought\t\r \u00a0to\t\r \u00a0activate\t\r \u00a0RhoG\t\r \u00a0indirectly\t\r \u00a0via\t\r \u00a0its\t\r \u00a0activity\t\r \u00a0on\t\r \u00a0RhoG\t\r \u00a0through\t\r \u00a0SH3-\u00ad\u2010containing\t\r \u00a0GEF\t\r \u00a0(SGEF)(45,\t\r \u00a0168,\t\r \u00a0185-\u00ad\u2010187).\t\r \u00a0Redundancy\t\r \u00a0has\t\r \u00a0been\t\r \u00a0observed\t\r \u00a0among\t\r \u00a0these\t\r \u00a0effectors,\t\r \u00a0as\t\r \u00a0Salmonella\t\r \u00a0mutated\t\r \u00a0in\t\r \u00a0any\t\r \u00a0individual\t\r \u00a0effector\t\r \u00a0are\t\r \u00a0still\t\r \u00a0invasive.\t\r \u00a0However,\t\r \u00a0mutants\t\r \u00a0lacking\t\r \u00a0all\t\r \u00a0three\t\r \u00a0are\t\r \u00a0completely\t\r \u00a0abrogated\t\r \u00a0for\t\r \u00a0invasion\t\r \u00a0into\t\r \u00a0non-\u00ad\u2010phagocytic\t\r \u00a0cells(187).\t\r \u00a0Two\t\r \u00a0other\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0effectors,\t\r \u00a0SipA\t\r \u00a0and\t\r \u00a0SipC\t\r \u00a0(also\t\r \u00a0known\t\r \u00a0as\t\r \u00a0SspA\t\r \u00a0and\t\r \u00a0SspC),\t\r \u00a0also\t\r \u00a0promote\t\r \u00a0bacterial\t\r \u00a0internalization\t\r \u00a0by\t\r \u00a0regulating\t\r \u00a0actin\t\r \u00a0dynamics.\t\r \u00a0SipA\t\r \u00a0induces\t\r \u00a0actin\t\r \u00a0polymerization\t\r \u00a0at\t\r \u00a0sites\t\r \u00a0of\t\r \u00a0invasion\t\r \u00a0by\t\r \u00a0decreasing\t\r \u00a0the\t\r \u00a0critical\t\r \u00a0concentration\t\r \u00a0and\t\r \u00a0increasing\t\r \u00a0the\t\r \u00a0stability\t\r \u00a0of\t\r \u00a0F-\u00ad\u2010actin(145,\t\r \u00a0188).\t\r \u00a0SipC,\t\r \u00a0which\t\r \u00a0is\t\r \u00a0also\t\r \u00a0a\t\r \u00a0translocon,\t\r \u00a0has\t\r \u00a0a\t\r \u00a0cytosolic\t\r \u00a0domain\t\r \u00a0that\t\r \u00a0can\t\r \u00a0both\t\r \u00a0nucleate\t\r \u00a0and\t\r \u00a0bundle\t\r \u00a0actin\t\r \u00a0filaments\t\r \u00a0into\t\r \u00a0cables(154).\t\r \u00a0Co-\u00ad\u2010operation\t\r \u00a0between\t\r \u00a0these\t\r \u00a0two\t\r \u00a0effectors\t\r \u00a0has\t\r \u00a0also\t\r \u00a0been\t\r \u00a0shown,\t\r \u00a0as\t\r \u00a0SipA\t\r \u00a0enhances\t\r \u00a0nucleation\t\r \u00a0and\t\r \u00a0bundling\t\r \u00a0by\t\r \u00a0SipC(158).\t\r \u00a0However,\t\r \u00a0SipA\t\r \u00a0and\t\r \u00a0SipC\t\r \u00a0cannot\t\r \u00a0induce\t\r \u00a0internalization\t\r \u00a0in\t\r \u00a0the\t\r \u00a0absence\t\r \u00a0of\t\r \u00a0SopB\t\r \u00a0and\/or\t\r \u00a0SopE\/E2(187).\t\r \u00a0\t\r \u00a0 1.1.5.4.2\t\r \u00a0Innate\t\r \u00a0immune\t\r \u00a0signaling\t\r \u00a0Several\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0effectors\t\r \u00a0are\t\r \u00a0known\t\r \u00a0to\t\r \u00a0actively\t\r \u00a0induce\t\r \u00a0innate\t\r \u00a0immune\t\r \u00a0signaling\t\r \u00a0cascades\t\r \u00a0in\t\r \u00a0epithelial\t\r \u00a0cells\t\r \u00a0during\t\r \u00a0invasion,\t\r \u00a0which\t\r \u00a0results\t\r \u00a0in\t\r \u00a0the\t\r \u00a0production\t\r \u00a0of\t\r \u00a0cytokines\t\r \u00a0(i.e.\t\r \u00a0IL-\u00ad\u20108)\t\r \u00a0and\t\r \u00a0recruitment\t\r \u00a0of\t\r \u00a0immune\t\r \u00a0cells\t\r \u00a0such\t\r \u00a0as\t\r \u00a0PMNs\t\r \u00a0(Figure\t\r \u00a01.2)(31-\u00ad\u201033,\t\r \u00a0189).\t\r \u00a0While\t\r \u00a0previously\t\r \u00a0suggested\t\r \u00a0that\t\r \u00a0induction\t\r \u00a0of\t\r \u00a0inflammation\t\r \u00a0was\t\r \u00a0beneficial\t\r \u00a0to\t\r \u00a0Salmonella\t\r \u00a0growth\t\r \u00a0and\t\r \u00a0transmission,\t\r \u00a0it\t\r \u00a0has\t\r \u00a0recently\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0that\t\r \u00a0 \t\r \u00a0\t\r \u00a0 19\t\r \u00a0 Salmonella\t\r \u00a0use\t\r \u00a0tetrathionate\t\r \u00a0derived\t\r \u00a0from\t\r \u00a0reactive\t\r \u00a0oxygen\t\r \u00a0species\t\r \u00a0as\t\r \u00a0an\t\r \u00a0electron\t\r \u00a0acceptor\t\r \u00a0for\t\r \u00a0respiration(190,\t\r \u00a0191).\t\r \u00a0This\t\r \u00a0gives\t\r \u00a0them\t\r \u00a0an\t\r \u00a0advantage\t\r \u00a0over\t\r \u00a0commensal\t\r \u00a0microbes,\t\r \u00a0and\t\r \u00a0thus,\t\r \u00a0has\t\r \u00a0established\t\r \u00a0these\t\r \u00a0signaling\t\r \u00a0cascades\t\r \u00a0as\t\r \u00a0central\t\r \u00a0to\t\r \u00a0pathogenesis\t\r \u00a0by\t\r \u00a0promoting\t\r \u00a0Salmonella\t\r \u00a0replication\t\r \u00a0in\t\r \u00a0the\t\r \u00a0intestinal\t\r \u00a0lumen.\t\r \u00a0Similar\t\r \u00a0to\t\r \u00a0invasion,\t\r \u00a0these\t\r \u00a0effects\t\r \u00a0are\t\r \u00a0mediated\t\r \u00a0through\t\r \u00a0SopB\/E\t\r \u00a0and\t\r \u00a0E2.\t\r \u00a0These\t\r \u00a0effectors\t\r \u00a0activate\t\r \u00a0Cdc42\t\r \u00a0to\t\r \u00a0induce\t\r \u00a0Mitogen-\u00ad\u2010activated\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a0(MAPK)\t\r \u00a0signaling\t\r \u00a0within\t\r \u00a0the\t\r \u00a0JNK,\t\r \u00a0p38,\t\r \u00a0and\t\r \u00a0ERK1\/2\t\r \u00a0signaling\t\r \u00a0cascades\t\r \u00a0leading\t\r \u00a0to\t\r \u00a0activation\t\r \u00a0of\t\r \u00a0transcription\t\r \u00a0factors\t\r \u00a0Activator\t\r \u00a0protein-\u00ad\u20101\t\r \u00a0(AP-\u00ad\u20101)\t\r \u00a0and\t\r \u00a0Nuclear\t\r \u00a0factor-\u00ad\u2010kB\t\r \u00a0(NF-\u00ad\u2010\u03baB)(45).\t\r \u00a0Both\t\r \u00a0SopE\t\r \u00a0and\t\r \u00a0E2\t\r \u00a0activate\t\r \u00a0Cdc42\t\r \u00a0directly(174).\t\r \u00a0However,\t\r \u00a0how\t\r \u00a0SopB\t\r \u00a0induces\t\r \u00a0Cdc42\t\r \u00a0activation\t\r \u00a0is\t\r \u00a0currently\t\r \u00a0unknown.\t\r \u00a0SopB\t\r \u00a0also\t\r \u00a0activates\t\r \u00a0Akt,\t\r \u00a0likely\t\r \u00a0through\t\r \u00a0phosphoinositide\t\r \u00a0fluxes,\t\r \u00a0which\t\r \u00a0leads\t\r \u00a0to\t\r \u00a0activation\t\r \u00a0of\t\r \u00a0p21-\u00ad\u2010activated\t\r \u00a0kinase\t\r \u00a04\t\r \u00a0(PAK4)(66,\t\r \u00a0163).\t\r \u00a0In\t\r \u00a0addition,\t\r \u00a0SipA\t\r \u00a0has\t\r \u00a0also\t\r \u00a0been\t\r \u00a0reported\t\r \u00a0to\t\r \u00a0induce\t\r \u00a0signaling\t\r \u00a0in\t\r \u00a0epithelial\t\r \u00a0cells\t\r \u00a0to\t\r \u00a0promote\t\r \u00a0PMN\t\r \u00a0transepithelial\t\r \u00a0migration(147).\t\r \u00a0This\t\r \u00a0is\t\r \u00a0thought\t\r \u00a0to\t\r \u00a0occur\t\r \u00a0through\t\r \u00a0a\t\r \u00a0Protein\t\r \u00a0kinase\t\r \u00a0C\t\r \u00a0alpha\t\r \u00a0(PKC\u03b1)-\u00ad\u2010dependent\t\r \u00a0pathway,\t\r \u00a0also\t\r \u00a0involving\t\r \u00a0the\t\r \u00a0ADP-\u00ad\u2010ribosylation\t\r \u00a0factor\t\r \u00a06\t\r \u00a0(ARF6)\t\r \u00a0and\t\r \u00a0phospholipase\t\r \u00a0D(192,\t\r \u00a0193).\t\r \u00a0Interestingly,\t\r \u00a0extracellular\t\r \u00a0addition\t\r \u00a0of\t\r \u00a0SipA\t\r \u00a0was\t\r \u00a0sufficient\t\r \u00a0to\t\r \u00a0induce\t\r \u00a0these\t\r \u00a0responses.\t\r \u00a0SipA\t\r \u00a0translocated\t\r \u00a0into\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0has\t\r \u00a0also\t\r \u00a0been\t\r \u00a0reported\t\r \u00a0to\t\r \u00a0induce\t\r \u00a0expression\t\r \u00a0of\t\r \u00a0CXC-\u00ad\u2010chemokines\t\r \u00a0through\t\r \u00a0JUN\t\r \u00a0and\t\r \u00a0p38(194).\t\r \u00a0However,\t\r \u00a0in\t\r \u00a0each\t\r \u00a0of\t\r \u00a0these\t\r \u00a0cases\t\r \u00a0the\t\r \u00a0direct\t\r \u00a0target(s)\t\r \u00a0of\t\r \u00a0SipA\t\r \u00a0are\t\r \u00a0unknown.\t\r \u00a0 \t\r \u00a0\t\r \u00a0 20\t\r \u00a0 Figure\t\r \u00a01.2\t\r \u00a0Activity\t\r \u00a0flow\t\r \u00a0diagram\t\r \u00a0of\t\r \u00a0phosphorylation\t\r \u00a0based\t\r \u00a0signaling\t\r \u00a0cascades\t\r \u00a0initiated\t\r \u00a0by\t\r \u00a0 T3SS-\u00ad\u20101\t\r \u00a0effectors\t\r \u00a0 \t\r \u00a0\t\r \u00a0 Salmonella\t\r \u00a0effectors\t\r \u00a0are\t\r \u00a0shown\t\r \u00a0in\t\r \u00a0blue,\t\r \u00a0and\t\r \u00a0proteins\t\r \u00a0phosphorylated\t\r \u00a0during\t\r \u00a0infection\t\r \u00a0are\t\r \u00a0shown\t\r \u00a0in\t\r \u00a0red.\t\r \u00a0Biological\t\r \u00a0activities\t\r \u00a0are\t\r \u00a0shown\t\r \u00a0in\t\r \u00a0boxes,\t\r \u00a0and\t\r \u00a0the\t\r \u00a0influences\t\r \u00a0of\t\r \u00a0each\t\r \u00a0biological\t\r \u00a0activity\t\r \u00a0are\t\r \u00a0indicated\t\r \u00a0by\t\r \u00a0arcs\t\r \u00a0(lines)\t\r \u00a0directed\t\r \u00a0at\t\r \u00a0subsequent\t\r \u00a0biological\t\r \u00a0activities\t\r \u00a0or\t\r \u00a0at\t\r \u00a0phenotypes\t\r \u00a0(hexagonal\t\r \u00a0boxes).\t\r \u00a0Positive\t\r \u00a0influences\t\r \u00a0are\t\r \u00a0indicated\t\r \u00a0by\t\r \u00a0arrowheads\t\r \u00a0on\t\r \u00a0an\t\r \u00a0arc,\t\r \u00a0negative\t\r \u00a0influences\t\r \u00a0by\t\r \u00a0a\t\r \u00a0short\t\r \u00a0perpendicular\t\r \u00a0line\t\r \u00a0at\t\r \u00a0the\t\r \u00a0end\t\r \u00a0of\t\r \u00a0an\t\r \u00a0arc,\t\r \u00a0and\t\r \u00a0unknown\t\r \u00a0influence\t\r \u00a0is\t\r \u00a0shown\t\r \u00a0by\t\r \u00a0a\t\r \u00a0diamond\t\r \u00a0shape\t\r \u00a0at\t\r \u00a0the\t\r \u00a0end\t\r \u00a0of\t\r \u00a0an\t\r \u00a0arc\t\r \u00a0(i.e.\t\r \u00a0SspH1).\t\r \u00a0A\t\r \u00a0necessary\t\r \u00a0influence\t\r \u00a0(one\t\r \u00a0required\t\r \u00a0for\t\r \u00a0the\t\r \u00a0subsequent\t\r \u00a0biological\t\r \u00a0activity)\t\r \u00a0is\t\r \u00a0indicated\t\r \u00a0by\t\r \u00a0a\t\r \u00a0perpendicular\t\r \u00a0line\t\r \u00a0followed\t\r \u00a0by\t\r \u00a0an\t\r \u00a0arrowhead\t\r \u00a0(i.e.\t\r \u00a0IL-\u00ad\u20108).\t\r \u00a0Modifiers\t\r \u00a0added\t\r \u00a0to\t\r \u00a0arcs\t\r \u00a0include\t\r \u00a0Boolean\t\r \u00a0terms\t\r \u00a0and\t\r \u00a0the\t\r \u00a0greek\t\r \u00a0letter\t\r \u00a0'tau',\t\r \u00a0which\t\r \u00a0indicates\t\r \u00a0that\t\r \u00a0the\t\r \u00a0biological\t\r \u00a0activity\t\r \u00a0is\t\r \u00a0delayed.\t\r \u00a0This\t\r \u00a0diagram\t\r \u00a0is\t\r \u00a0designed\t\r \u00a0to\t\r \u00a0conform\t\r \u00a0to\t\r \u00a0the\t\r \u00a0systems\t\r \u00a0biology\t\r \u00a0graphic\t\r \u00a0notation(195).\t\r \u00a0\t\r \u00a0 1.1.5.4.3\t\r \u00a0Reversal\t\r \u00a0of\t\r \u00a0cytoskeletal\t\r \u00a0and\t\r \u00a0signaling\t\r \u00a0responses\t\r \u00a0Several\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0effectors\t\r \u00a0are\t\r \u00a0also\t\r \u00a0known\t\r \u00a0to\t\r \u00a0down-\u00ad\u2010regulate\t\r \u00a0the\t\r \u00a0effects\t\r \u00a0initiated\t\r \u00a0by\t\r \u00a0SopB\/E\/E2\t\r \u00a0and\t\r \u00a0SipA\/C.\t\r \u00a0Shortly\t\r \u00a0after\t\r \u00a0invasion\t\r \u00a0by\t\r \u00a0Salmonella,\t\r \u00a0the\t\r \u00a0actin\t\r \u00a0cytoskeleton\t\r \u00a0of\t\r \u00a0infected\t\r \u00a0epithelial\t\r \u00a0cells\t\r \u00a0regains\t\r \u00a0its\t\r \u00a0normal\t\r \u00a0architecture(17).\t\r \u00a0This\t\r \u00a0is\t\r \u00a0mediated\t\r \u00a0by\t\r \u00a0the\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0effector\t\r \u00a0SptP,\t\r \u00a0which\t\r \u00a0is\t\r \u00a0a\t\r \u00a0GTPase-\u00ad\u2010activating\t\r \u00a0protein\t\r \u00a0(GAP)\t\r \u00a0for\t\r \u00a0Cdc42\t\r \u00a0and\t\r \u00a0Rac1(176).\t\r \u00a0This\t\r \u00a0effect\t\r \u00a0is\t\r \u00a0also\t\r \u00a0regulated\t\r \u00a0by\t\r \u00a0the\t\r \u00a0varying\t\r \u00a0stabilities\t\r \u00a0of\t\r \u00a0these\t\r \u00a0proteins\t\r \u00a0in\t\r \u00a0the\t\r \u00a0host\t\r \u00a0cytosol,\t\r \u00a0as\t\r \u00a0SptP\t\r \u00a0has\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0persist\t\r \u00a0in\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0much\t\r \u00a0 \t\r \u00a0\t\r \u00a0 21\t\r \u00a0 longer\t\r \u00a0than\t\r \u00a0SopE\/E2,\t\r \u00a0which\t\r \u00a0are\t\r \u00a0rapidly\t\r \u00a0degraded\t\r \u00a0by\t\r \u00a0the\t\r \u00a0proteasome(196).\t\r \u00a0Aside\t\r \u00a0from\t\r \u00a0cytoskeletal\t\r \u00a0arrangements,\t\r \u00a0several\t\r \u00a0effectors\t\r \u00a0have\t\r \u00a0also\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0down-\u00ad\u2010regulate\t\r \u00a0MAPK\t\r \u00a0signaling\t\r \u00a0downstream\t\r \u00a0of\t\r \u00a0Cdc42.\t\r \u00a0In\t\r \u00a0addition\t\r \u00a0to\t\r \u00a0its\t\r \u00a0activity\t\r \u00a0as\t\r \u00a0a\t\r \u00a0GAP,\t\r \u00a0SptP\t\r \u00a0is\t\r \u00a0also\t\r \u00a0a\t\r \u00a0protein\t\r \u00a0tyrosine\t\r \u00a0phosphatase\t\r \u00a0(PTP)(175).\t\r \u00a0Thus,\t\r \u00a0it\t\r \u00a0has\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0down-\u00ad\u2010regulate\t\r \u00a0MAPK\t\r \u00a0signaling\t\r \u00a0cascades\t\r \u00a0through\t\r \u00a0its\t\r \u00a0GAP\t\r \u00a0activity\t\r \u00a0on\t\r \u00a0Cdc42,\t\r \u00a0and\t\r \u00a0also\t\r \u00a0through\t\r \u00a0RAF\t\r \u00a0proto-\u00ad\u2010oncogene\t\r \u00a0serine\/threonine-\u00ad\u2010protein\t\r \u00a0kinase\t\r \u00a0(Raf1)\t\r \u00a0inactivation\t\r \u00a0which\t\r \u00a0is\t\r \u00a0dependent\t\r \u00a0on\t\r \u00a0both\t\r \u00a0its\t\r \u00a0GAP\t\r \u00a0and\t\r \u00a0tyrosine\t\r \u00a0phosphatase\t\r \u00a0activities(176,\t\r \u00a0178).\t\r \u00a0Two\t\r \u00a0other\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0effectors\t\r \u00a0also\t\r \u00a0inhibit\t\r \u00a0innate\t\r \u00a0immune\t\r \u00a0signaling.\t\r \u00a0SspH1\t\r \u00a0localizes\t\r \u00a0to\t\r \u00a0the\t\r \u00a0nucleus\t\r \u00a0and\t\r \u00a0binds\t\r \u00a0Serine\/threonine\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a0N1\t\r \u00a0(PKN1)\t\r \u00a0to\t\r \u00a0inhibit\t\r \u00a0NF-\u00ad\u2010\u03baB\t\r \u00a0dependent\t\r \u00a0gene\t\r \u00a0expression,\t\r \u00a0and\t\r \u00a0AvrA\t\r \u00a0inhibits\t\r \u00a0Dual\t\r \u00a0specificity\t\r \u00a0mitogen-\u00ad\u2010activated\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a0kinase\t\r \u00a07\t\r \u00a0(MEK7)\t\r \u00a0through\t\r \u00a0acetylation\t\r \u00a0at\t\r \u00a0serine\t\r \u00a0and\t\r \u00a0threonine\t\r \u00a0residues,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0NF-\u00ad\u2010\u03baB\t\r \u00a0activation\t\r \u00a0by\t\r \u00a0preventing\t\r \u00a0degradation\t\r \u00a0of\t\r \u00a0NF-\u00ad\u2010kappa-\u00ad\u2010B\t\r \u00a0inhibitor\t\r \u00a0alpha\t\r \u00a0(I-\u00ad\u2010\u03baB\u03b1)(143,\t\r \u00a0144,\t\r \u00a0182,\t\r \u00a0183)).\t\r \u00a0\t\r \u00a0 1.1.5.4.4\t\r \u00a0Formation\t\r \u00a0and\t\r \u00a0trafficking\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV\t\r \u00a0Although\t\r \u00a0generally\t\r \u00a0attributed\t\r \u00a0to\t\r \u00a0SPI-\u00ad\u20102\t\r \u00a0effectors,\t\r \u00a0several\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0effectors\t\r \u00a0have\t\r \u00a0also\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0regulate\t\r \u00a0formation\t\r \u00a0and\t\r \u00a0trafficking\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV,\t\r \u00a0and\t\r \u00a0to\t\r \u00a0promote\t\r \u00a0bacterial\t\r \u00a0replication\t\r \u00a0within\t\r \u00a0host\t\r \u00a0cells.\t\r \u00a0SopB\t\r \u00a0initially\t\r \u00a0localizes\t\r \u00a0to\t\r \u00a0the\t\r \u00a0plasma\t\r \u00a0membrane\t\r \u00a0where\t\r \u00a0it\t\r \u00a0regulates\t\r \u00a0macropinocytosis\t\r \u00a0and\t\r \u00a0formation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV.\t\r \u00a0Through\t\r \u00a0its\t\r \u00a0phosphatase\t\r \u00a0activity,\t\r \u00a0SopB\t\r \u00a0depletes\t\r \u00a0PI(4,5)P2\t\r \u00a0from\t\r \u00a0invaginations\t\r \u00a0in\t\r \u00a0the\t\r \u00a0plasma\t\r \u00a0membrane,\t\r \u00a0which\t\r \u00a0is\t\r \u00a0required\t\r \u00a0for\t\r \u00a0fission\t\r \u00a0and\t\r \u00a0formation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0macropinosome(49).\t\r \u00a0Several\t\r \u00a0vesicles\t\r \u00a0containing\t\r \u00a0VAMP8\t\r \u00a0are\t\r \u00a0also\t\r \u00a0recruited\t\r \u00a0to\t\r \u00a0Salmonella-\u00ad\u2010induced\t\r \u00a0membrane\t\r \u00a0ruffles\t\r \u00a0in\t\r \u00a0a\t\r \u00a0SopB\t\r \u00a0dependent\t\r \u00a0manner,\t\r \u00a0which\t\r \u00a0provide\t\r \u00a0a\t\r \u00a0membrane\t\r \u00a0source\t\r \u00a0 \t\r \u00a0\t\r \u00a0 22\t\r \u00a0 for\t\r \u00a0macropinosome\t\r \u00a0formation(50).\t\r \u00a0Ultimately,\t\r \u00a0infection\t\r \u00a0with\t\r \u00a0a\t\r \u00a0\u0394sopB\t\r \u00a0strain\t\r \u00a0results\t\r \u00a0in\t\r \u00a0smaller,\t\r \u00a0shorter\t\r \u00a0lived\t\r \u00a0macropinosomes,\t\r \u00a0while\t\r \u00a0those\t\r \u00a0formed\t\r \u00a0during\t\r \u00a0WT\t\r \u00a0infection\t\r \u00a0are\t\r \u00a0large,\t\r \u00a0persist\t\r \u00a0for\t\r \u00a0up\t\r \u00a0to\t\r \u00a090\t\r \u00a0min,\t\r \u00a0and\t\r \u00a0fuse\t\r \u00a0with\t\r \u00a0multiple\t\r \u00a0PI3P\t\r \u00a0enriched\t\r \u00a0vesicles\t\r \u00a0shortly\t\r \u00a0after\t\r \u00a0formation(57).\t\r \u00a0Once\t\r \u00a0inside\t\r \u00a0host\t\r \u00a0cells,\t\r \u00a0SopB\t\r \u00a0is\t\r \u00a0multimonoubiquitylated\t\r \u00a0and\t\r \u00a0re-\u00ad\u2010localizes\t\r \u00a0from\t\r \u00a0the\t\r \u00a0plasma\t\r \u00a0membrane\t\r \u00a0to\t\r \u00a0the\t\r \u00a0SCV(197,\t\r \u00a0198).\t\r \u00a0Here\t\r \u00a0it\t\r \u00a0is\t\r \u00a0required\t\r \u00a0for\t\r \u00a0recruitment\t\r \u00a0of\t\r \u00a0SNX-\u00ad\u20101\t\r \u00a0and\t\r \u00a0-\u00ad\u20103\t\r \u00a0to\t\r \u00a0SCVs(59,\t\r \u00a060).\t\r \u00a0SNX-\u00ad\u20101\t\r \u00a0and\t\r \u00a0-\u00ad\u20103\t\r \u00a0bind\t\r \u00a0phosphoinositides,\t\r \u00a0and\t\r \u00a0are\t\r \u00a0required\t\r \u00a0for\t\r \u00a0early\t\r \u00a0tubulation\t\r \u00a0and\t\r \u00a0remodeling\t\r \u00a0of\t\r \u00a0the\t\r \u00a0nascent\t\r \u00a0SCV,\t\r \u00a0reducing\t\r \u00a0its\t\r \u00a0size.\t\r \u00a0SopB\u2019s\t\r \u00a0phosphatase\t\r \u00a0activity\t\r \u00a0is\t\r \u00a0required\t\r \u00a0for\t\r \u00a0Rab5\t\r \u00a0and\t\r \u00a0Vps34\t\r \u00a0recruitment\t\r \u00a0to\t\r \u00a0the\t\r \u00a0SCV,\t\r \u00a0the\t\r \u00a0later\t\r \u00a0of\t\r \u00a0which\t\r \u00a0forms\t\r \u00a0PI3P(58).\t\r \u00a0It\t\r \u00a0also\t\r \u00a0depletes\t\r \u00a0PI(4,5)P2\t\r \u00a0and\t\r \u00a0phosphatidyl\t\r \u00a0serine\t\r \u00a0from\t\r \u00a0the\t\r \u00a0SCV,\t\r \u00a0which\t\r \u00a0decreases\t\r \u00a0the\t\r \u00a0electrostatic\t\r \u00a0surface\t\r \u00a0charge\t\r \u00a0and\t\r \u00a0causes\t\r \u00a0dissociation\t\r \u00a0of\t\r \u00a0several\t\r \u00a0host\t\r \u00a0trafficking\t\r \u00a0proteins\t\r \u00a0to\t\r \u00a0inhibit\t\r \u00a0SCV-\u00ad\u2010lysosome\t\r \u00a0fusion(199).\t\r \u00a0SopB\t\r \u00a0has\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0regulate\t\r \u00a0myosin\t\r \u00a0II\t\r \u00a0activity\t\r \u00a0for\t\r \u00a0perinuclear\t\r \u00a0positioning\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV(70).\t\r \u00a0When\t\r \u00a0transfected\t\r \u00a0in\t\r \u00a0host\t\r \u00a0cells,\t\r \u00a0SopB\t\r \u00a0can\t\r \u00a0inhibit\t\r \u00a0trafficking\t\r \u00a0of\t\r \u00a0the\t\r \u00a0EGFR\t\r \u00a0to\t\r \u00a0lysosomes,\t\r \u00a0and\t\r \u00a0during\t\r \u00a0infection\t\r \u00a0it\t\r \u00a0is\t\r \u00a0required\t\r \u00a0for\t\r \u00a0SCV\t\r \u00a0maturation\t\r \u00a0and\t\r \u00a0intracellular\t\r \u00a0bacterial\t\r \u00a0growth,\t\r \u00a0and\t\r \u00a0can\t\r \u00a0persist\t\r \u00a0in\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0for\t\r \u00a0more\t\r \u00a0than\t\r \u00a012\t\r \u00a0h\t\r \u00a0post\t\r \u00a0invasion(57,\t\r \u00a0200,\t\r \u00a0201).\t\r \u00a0Similar\t\r \u00a0to\t\r \u00a0SopB,\t\r \u00a0SipC,\t\r \u00a0SopD,\t\r \u00a0SopE,\t\r \u00a0SptP\t\r \u00a0and\t\r \u00a0SipA\t\r \u00a0have\t\r \u00a0also\t\r \u00a0be\t\r \u00a0reported\t\r \u00a0to\t\r \u00a0affect\t\r \u00a0formation\t\r \u00a0and\t\r \u00a0trafficking\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV.\t\r \u00a0Both\t\r \u00a0SipC\t\r \u00a0and\t\r \u00a0SopD\t\r \u00a0are\t\r \u00a0thought\t\r \u00a0to\t\r \u00a0act\t\r \u00a0synergistically\t\r \u00a0with\t\r \u00a0SopB.\t\r \u00a0SipC\t\r \u00a0enhances\t\r \u00a0invasion\t\r \u00a0through\t\r \u00a0clustering\t\r \u00a0of\t\r \u00a0PI(4,5)P2\t\r \u00a0at\t\r \u00a0the\t\r \u00a0plasma\t\r \u00a0membrane,\t\r \u00a0while\t\r \u00a0SopD\t\r \u00a0localizes\t\r \u00a0to\t\r \u00a0invasion\t\r \u00a0sites\t\r \u00a0and\t\r \u00a0contributes\t\r \u00a0to\t\r \u00a0membrane\t\r \u00a0fission\t\r \u00a0and\t\r \u00a0macropinosome\t\r \u00a0formation(167,\t\r \u00a0202).\t\r \u00a0SopE\t\r \u00a0acts\t\r \u00a0as\t\r \u00a0a\t\r \u00a0GEF\t\r \u00a0for\t\r \u00a0Rab5\t\r \u00a0and\t\r \u00a0regulates\t\r \u00a0its\t\r \u00a0recruitment\t\r \u00a0to\t\r \u00a0SCVs,\t\r \u00a0which\t\r \u00a0promotes\t\r \u00a0trafficking\t\r \u00a0to\t\r \u00a0early\t\r \u00a0 \t\r \u00a0\t\r \u00a0 23\t\r \u00a0 endosomes\t\r \u00a0but\t\r \u00a0not\t\r \u00a0lysosmal\t\r \u00a0compartments(170,\t\r \u00a0171).\t\r \u00a0Alternatively,\t\r \u00a0SptP\t\r \u00a0persists\t\r \u00a0in\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0for\t\r \u00a0at\t\r \u00a0least\t\r \u00a08\t\r \u00a0h\t\r \u00a0post\t\r \u00a0invasion(179).\t\r \u00a0Its\t\r \u00a0PTP\t\r \u00a0activity\t\r \u00a0promotes\t\r \u00a0bacterial\t\r \u00a0replication\t\r \u00a0through\t\r \u00a0dephosphorylation\t\r \u00a0of\t\r \u00a0host\t\r \u00a0Transitional\t\r \u00a0endoplasmic\t\r \u00a0reticulum\t\r \u00a0ATPase\t\r \u00a0(VCP),\t\r \u00a0which\t\r \u00a0is\t\r \u00a0required\t\r \u00a0for\t\r \u00a0the\t\r \u00a0formation\t\r \u00a0of\t\r \u00a0SIFs.\t\r \u00a0Similarly,\t\r \u00a0SipA\t\r \u00a0has\t\r \u00a0also\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0persist\t\r \u00a0in\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0long\t\r \u00a0after\t\r \u00a0invasion,\t\r \u00a0and\t\r \u00a0to\t\r \u00a0co-\u00ad\u2010operate\t\r \u00a0with\t\r \u00a0the\t\r \u00a0SPI-\u00ad\u20102\t\r \u00a0effector\t\r \u00a0SifA\t\r \u00a0for\t\r \u00a0positioning\t\r \u00a0and\t\r \u00a0trafficking\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0bacterial\t\r \u00a0replication\t\r \u00a0within\t\r \u00a0infected\t\r \u00a0cells(149).\t\r \u00a0\t\r \u00a0 1.1.5.4.5\t\r \u00a0Other\t\r \u00a0effects\t\r \u00a0of\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0effectors\t\r \u00a0Several\t\r \u00a0other\t\r \u00a0effects\t\r \u00a0have\t\r \u00a0been\t\r \u00a0attributed\t\r \u00a0to\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0effectors.\t\r \u00a0At\t\r \u00a0the\t\r \u00a0cell\t\r \u00a0periphery\t\r \u00a0these\t\r \u00a0include\t\r \u00a0modulation\t\r \u00a0of\t\r \u00a0chloride\t\r \u00a0ion\t\r \u00a0secretion,\t\r \u00a0nitric\t\r \u00a0oxide\t\r \u00a0production,\t\r \u00a0and\t\r \u00a0reduction\t\r \u00a0in\t\r \u00a0trans\t\r \u00a0epithelial\t\r \u00a0resistance\t\r \u00a0in\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0by\t\r \u00a0SopB,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0disruption\t\r \u00a0of\t\r \u00a0tight\t\r \u00a0junctions\t\r \u00a0by\t\r \u00a0SopB\/E\/E2\t\r \u00a0and\t\r \u00a0SipA(148,\t\r \u00a0164,\t\r \u00a0201).\t\r \u00a0Several\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0effectors\t\r \u00a0have\t\r \u00a0also\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0regulate\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0death.\t\r \u00a0These\t\r \u00a0include\t\r \u00a0SopB\t\r \u00a0and\t\r \u00a0Akt\t\r \u00a0dependent\t\r \u00a0anti-\u00ad\u2010apoptotic\t\r \u00a0signaling,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0caspase-\u00ad\u20101\t\r \u00a0induced\t\r \u00a0pyroptosis\t\r \u00a0in\t\r \u00a0macrophages\t\r \u00a0by\t\r \u00a0SipB,\t\r \u00a0and\t\r \u00a0also\t\r \u00a0by\t\r \u00a0SopE\t\r \u00a0downstream\t\r \u00a0of\t\r \u00a0Rac1\t\r \u00a0and\t\r \u00a0Cdc42(151,\t\r \u00a0165,\t\r \u00a0172).\t\r \u00a0Both\t\r \u00a0SlrP\t\r \u00a0and\t\r \u00a0SopA\t\r \u00a0are\t\r \u00a0E3\t\r \u00a0ubiquitin\t\r \u00a0ligases.\t\r \u00a0SlrP\t\r \u00a0has\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0promote\t\r \u00a0cell\t\r \u00a0death\t\r \u00a0in\t\r \u00a0epithelial\t\r \u00a0cells\t\r \u00a0through\t\r \u00a0thioredoxin\t\r \u00a0degradation,\t\r \u00a0while\t\r \u00a0also\t\r \u00a0targeting\t\r \u00a0ER-\u00ad\u2010associated\t\r \u00a0dnaJ\t\r \u00a0protein\t\r \u00a03\t\r \u00a0(ERdj3)\t\r \u00a0at\t\r \u00a0the\t\r \u00a0endoplasmic\t\r \u00a0reticulum(180,\t\r \u00a0181).\t\r \u00a0Currently\t\r \u00a0undefined\t\r \u00a0targets\t\r \u00a0of\t\r \u00a0SopA\t\r \u00a0have\t\r \u00a0been\t\r \u00a0reported\t\r \u00a0to\t\r \u00a0decrease\t\r \u00a0transepithelial\t\r \u00a0migration\t\r \u00a0of\t\r \u00a0PMNs(162).\t\r \u00a0Interestingly,\t\r \u00a0SipA\t\r \u00a0has\t\r \u00a0recently\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0activate\t\r \u00a0and\t\r \u00a0subsequently\t\r \u00a0be\t\r \u00a0processed\t\r \u00a0by\t\r \u00a0caspase-\u00ad\u20103(150).\t\r \u00a0\t\r \u00a0 \t\r \u00a0\t\r \u00a0 24\t\r \u00a0 1.1.5.5\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0chaperones\t\r \u00a0Given\t\r \u00a0the\t\r \u00a0complex\t\r \u00a0assembly\t\r \u00a0process\t\r \u00a0and\t\r \u00a0limited\t\r \u00a0number\t\r \u00a0of\t\r \u00a0specific\t\r \u00a0effectors\t\r \u00a0and\t\r \u00a0translocons\t\r \u00a0destined\t\r \u00a0for\t\r \u00a0host\t\r \u00a0cells,\t\r \u00a0specificity\t\r \u00a0in\t\r \u00a0substrate\t\r \u00a0selection\t\r \u00a0for\t\r \u00a0secretion\t\r \u00a0through\t\r \u00a0the\t\r \u00a0needle\t\r \u00a0complex\t\r \u00a0is\t\r \u00a0very\t\r \u00a0important.\t\r \u00a0It\t\r \u00a0is\t\r \u00a0currently\t\r \u00a0unclear\t\r \u00a0how\t\r \u00a0this\t\r \u00a0is\t\r \u00a0achieved.\t\r \u00a0Most\t\r \u00a0secreted\t\r \u00a0T3SS\t\r \u00a0proteins\t\r \u00a0contain\t\r \u00a0a\t\r \u00a0secretion\t\r \u00a0signal\t\r \u00a0within\t\r \u00a0their\t\r \u00a0first\t\r \u00a020-\u00ad\u201030\t\r \u00a0residues.\t\r \u00a0However,\t\r \u00a0these\t\r \u00a0sequences\t\r \u00a0lack\t\r \u00a0conserved\t\r \u00a0features,\t\r \u00a0even\t\r \u00a0among\t\r \u00a0proteins\t\r \u00a0of\t\r \u00a0the\t\r \u00a0same\t\r \u00a0T3SS\t\r \u00a0and\t\r \u00a0it\t\r \u00a0is\t\r \u00a0unclear\t\r \u00a0how\t\r \u00a0or\t\r \u00a0if\t\r \u00a0they\t\r \u00a0dictate\t\r \u00a0substrate\t\r \u00a0specificity(203).\t\r \u00a0\t\r \u00a0An\t\r \u00a0additional\t\r \u00a0layer\t\r \u00a0of\t\r \u00a0specificity\t\r \u00a0is\t\r \u00a0thought\t\r \u00a0to\t\r \u00a0be\t\r \u00a0achieved\t\r \u00a0by\t\r \u00a0chaperones.\t\r \u00a0These\t\r \u00a0are\t\r \u00a0small,\t\r \u00a0acidic,\t\r \u00a0dimeric\t\r \u00a0proteins\t\r \u00a0that\t\r \u00a0generally\t\r \u00a0bind\t\r \u00a0a\t\r \u00a050-\u00ad\u2010100\t\r \u00a0residue\t\r \u00a0region\t\r \u00a0located\t\r \u00a0immediately\t\r \u00a0downstream\t\r \u00a0of\t\r \u00a0the\t\r \u00a0N-\u00ad\u2010terminal\t\r \u00a0secretion\t\r \u00a0signal,\t\r \u00a0and\t\r \u00a0are\t\r \u00a0typically\t\r \u00a0encoded\t\r \u00a0next\t\r \u00a0to\t\r \u00a0or\t\r \u00a0in\t\r \u00a0close\t\r \u00a0proximity\t\r \u00a0to\t\r \u00a0their\t\r \u00a0target(204-\u00ad\u2010207).\t\r \u00a0Most\t\r \u00a0T3SS\t\r \u00a0chaperones\t\r \u00a0bind\t\r \u00a0only\t\r \u00a0a\t\r \u00a0single\t\r \u00a0target,\t\r \u00a0and\t\r \u00a0structural\t\r \u00a0studies\t\r \u00a0have\t\r \u00a0shown\t\r \u00a0that\t\r \u00a0they\t\r \u00a0hold\t\r \u00a0the\t\r \u00a0target\t\r \u00a0protein\t\r \u00a0in\t\r \u00a0a\t\r \u00a0non-\u00ad\u2010globular\t\r \u00a0conformation\t\r \u00a0that\t\r \u00a0might\t\r \u00a0aid\t\r \u00a0unfolding\t\r \u00a0for\t\r \u00a0transport\t\r \u00a0through\t\r \u00a0the\t\r \u00a0needle\t\r \u00a0complex(208,\t\r \u00a0209).\t\r \u00a0\t\r \u00a0Several\t\r \u00a0chaperones\t\r \u00a0have\t\r \u00a0been\t\r \u00a0identified\t\r \u00a0for\t\r \u00a0both\t\r \u00a0translocons\t\r \u00a0and\t\r \u00a0effectors\t\r \u00a0secreted\t\r \u00a0through\t\r \u00a0T3SS-\u00ad\u20101.\t\r \u00a0These\t\r \u00a0include\t\r \u00a0SicA\t\r \u00a0which\t\r \u00a0is\t\r \u00a0a\t\r \u00a0chaperone\t\r \u00a0for\t\r \u00a0SipB\t\r \u00a0and\t\r \u00a0SipC(210),\t\r \u00a0SicP\t\r \u00a0which\t\r \u00a0is\t\r \u00a0a\t\r \u00a0chaperone\t\r \u00a0for\t\r \u00a0SptP(211),\t\r \u00a0SigE\t\r \u00a0which\t\r \u00a0is\t\r \u00a0a\t\r \u00a0chaperone\t\r \u00a0for\t\r \u00a0SopB(212,\t\r \u00a0213),\t\r \u00a0and\t\r \u00a0InvB\t\r \u00a0which\t\r \u00a0is\t\r \u00a0chaperone\t\r \u00a0for\t\r \u00a0SipA,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0SopE,\t\r \u00a0SopE2\t\r \u00a0and\t\r \u00a0SopA(214-\u00ad\u2010217).\t\r \u00a0These\t\r \u00a0chaperone-\u00ad\u2010effector\t\r \u00a0complexes\t\r \u00a0are\t\r \u00a0thought\t\r \u00a0to\t\r \u00a0interact\t\r \u00a0with\t\r \u00a0InvC\t\r \u00a0to\t\r \u00a0direct\t\r \u00a0entry\t\r \u00a0and\t\r \u00a0transport\t\r \u00a0through\t\r \u00a0the\t\r \u00a0needle\t\r \u00a0complex.\t\r \u00a0InvC\t\r \u00a0has\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0bind\t\r \u00a0the\t\r \u00a0SptP-\u00ad\u2010SicP\t\r \u00a0complex,\t\r \u00a0remove\t\r \u00a0the\t\r \u00a0effector\t\r \u00a0from\t\r \u00a0the\t\r \u00a0chaperone,\t\r \u00a0and\t\r \u00a0initiate\t\r \u00a0its\t\r \u00a0unfolding\t\r \u00a0and\t\r \u00a0transport\t\r \u00a0through\t\r \u00a0the\t\r \u00a0needle(218).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 25\t\r \u00a0 1.1.6\t\r \u00a0Salmonella-\u00ad\u2010pathogenicity\t\r \u00a0island\t\r \u00a02\t\r \u00a0 1.1.6.1\t\r \u00a0Regulation\t\r \u00a0of\t\r \u00a0SPI-\u00ad\u20102\t\r \u00a0 Salmonella-\u00ad\u2010pathogenicity\t\r \u00a0island\t\r \u00a02\t\r \u00a0is\t\r \u00a0required\t\r \u00a0for\t\r \u00a0replication\t\r \u00a0within\t\r \u00a0macrophages\t\r \u00a0and\t\r \u00a0systemic\t\r \u00a0infection\t\r \u00a0in\t\r \u00a0mice(29,\t\r \u00a093).\t\r \u00a0Within\t\r \u00a0SPI-\u00ad\u20102,\t\r \u00a0four\t\r \u00a0operons\t\r \u00a0are\t\r \u00a0thought\t\r \u00a0to\t\r \u00a0encode\t\r \u00a0components\t\r \u00a0of\t\r \u00a0the\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0apparatus,\t\r \u00a0effectors,\t\r \u00a0regulators,\t\r \u00a0and\t\r \u00a0chaperones(219).\t\r \u00a0These\t\r \u00a0are\t\r \u00a0designated\t\r \u00a0ssa,\t\r \u00a0sse,\t\r \u00a0ssr\t\r \u00a0and\t\r \u00a0ssc\t\r \u00a0genes\t\r \u00a0respectively.\t\r \u00a0SPI-\u00ad\u20102\t\r \u00a0is\t\r \u00a0expressed\t\r \u00a0under\t\r \u00a0conditions\t\r \u00a0such\t\r \u00a0as\t\r \u00a0low\t\r \u00a0Mg2+,\t\r \u00a0low\t\r \u00a0PO4-\u00ad\u2010\t\r \u00a0and\t\r \u00a0low\t\r \u00a0pH(220,\t\r \u00a0221).\t\r \u00a0Several\t\r \u00a0ancestral\t\r \u00a0regulators\t\r \u00a0such\t\r \u00a0as\t\r \u00a0the\t\r \u00a0OmpR\/EnvZ\t\r \u00a0and\t\r \u00a0PhoP\/PhoQ\t\r \u00a0two-\u00ad\u2010component\t\r \u00a0regulatory\t\r \u00a0systems,\t\r \u00a0and\t\r \u00a0the\t\r \u00a0regulator\t\r \u00a0protein\t\r \u00a0SlyA\t\r \u00a0have\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0activate\t\r \u00a0expression\t\r \u00a0and\t\r \u00a0translocation\t\r \u00a0of\t\r \u00a0SPI-\u00ad\u20102\t\r \u00a0genes,\t\r \u00a0although\t\r \u00a0regulation\t\r \u00a0by\t\r \u00a0PhoP\/PhoQ\t\r \u00a0is\t\r \u00a0controversial(222-\u00ad\u2010226).\t\r \u00a0These\t\r \u00a0activate\t\r \u00a0the\t\r \u00a0SPI-\u00ad\u20102\t\r \u00a0encoded\t\r \u00a0sensor\t\r \u00a0kinase\t\r \u00a0SsrA,\t\r \u00a0which\t\r \u00a0comprises\t\r \u00a0a\t\r \u00a0two-\u00ad\u2010component\t\r \u00a0regulatory\t\r \u00a0system\t\r \u00a0with\t\r \u00a0transcriptional\t\r \u00a0activator\t\r \u00a0SsrB(29,\t\r \u00a093).\t\r \u00a0SsrB\t\r \u00a0binds\t\r \u00a0to\t\r \u00a0and\t\r \u00a0activates\t\r \u00a0the\t\r \u00a0promoters\t\r \u00a0for\t\r \u00a0all\t\r \u00a0SPI-\u00ad\u20102\t\r \u00a0operons(227).\t\r \u00a0It\t\r \u00a0is\t\r \u00a0essential\t\r \u00a0for\t\r \u00a0expression\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SPI-\u00ad\u20102\t\r \u00a0encoded\t\r \u00a0T3SS\t\r \u00a0(T3SS-\u00ad\u20102),\t\r \u00a0and\t\r \u00a0all\t\r \u00a0effectors\t\r \u00a0encoded\t\r \u00a0within\t\r \u00a0and\t\r \u00a0outside\t\r \u00a0SPI-\u00ad\u20102(228).\t\r \u00a0\t\r \u00a0 1.1.6.2\t\r \u00a0T3SS-\u00ad\u20102\t\r \u00a0apparatus\t\r \u00a0Structural\t\r \u00a0characterizations\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0T3SS-\u00ad\u20102\t\r \u00a0are\t\r \u00a0far\t\r \u00a0lagging\t\r \u00a0that\t\r \u00a0of\t\r \u00a0T3SS-\u00ad\u20101.\t\r \u00a0However,\t\r \u00a0several\t\r \u00a0ssa\t\r \u00a0genes\t\r \u00a0within\t\r \u00a0SPI-\u00ad\u20102\t\r \u00a0are\t\r \u00a0homologous\t\r \u00a0to\t\r \u00a0components\t\r \u00a0of\t\r \u00a0other\t\r \u00a0T3SSs,\t\r \u00a0and\t\r \u00a0it\t\r \u00a0is\t\r \u00a0predicted\t\r \u00a0to\t\r \u00a0contain\t\r \u00a0all\t\r \u00a0the\t\r \u00a0components\t\r \u00a0required\t\r \u00a0for\t\r \u00a0a\t\r \u00a0functional\t\r \u00a0T3SS.\t\r \u00a0For\t\r \u00a0example,\t\r \u00a0ssaJ\t\r \u00a0and\t\r \u00a0ssaV\t\r \u00a0are\t\r \u00a0homologous\t\r \u00a0to\t\r \u00a0the\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0genes\t\r \u00a0prgK\t\r \u00a0and\t\r \u00a0invA(219).\t\r \u00a0Distinct\t\r \u00a0from\t\r \u00a0T3SS-\u00ad\u20101,\t\r \u00a0it\t\r \u00a0is\t\r \u00a0thought\t\r \u00a0that\t\r \u00a0only\t\r \u00a0a\t\r \u00a0single\t\r \u00a0T3SS-\u00ad\u20102\t\r \u00a0is\t\r \u00a0expressed\t\r \u00a0per\t\r \u00a0bacteria(229).\t\r \u00a0Also\t\r \u00a0unique\t\r \u00a0to\t\r \u00a0T3SS-\u00ad\u20102,\t\r \u00a0a\t\r \u00a0long\t\r \u00a0proteinaceous\t\r \u00a0 \t\r \u00a0\t\r \u00a0 26\t\r \u00a0 appendage\t\r \u00a0has\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0form\t\r \u00a0alongside\t\r \u00a0the\t\r \u00a0needle.\t\r \u00a0This\t\r \u00a0appendage\t\r \u00a0is\t\r \u00a0comprised\t\r \u00a0of\t\r \u00a0SseB,\t\r \u00a0forms\t\r \u00a0a\t\r \u00a0sheath\t\r \u00a0like\t\r \u00a0structure,\t\r \u00a0and\t\r \u00a0extends\t\r \u00a0from\t\r \u00a0the\t\r \u00a0inner\t\r \u00a0bacterial\t\r \u00a0membrane\t\r \u00a0to\t\r \u00a0the\t\r \u00a0tip\t\r \u00a0of\t\r \u00a0the\t\r \u00a0needle(229).\t\r \u00a0\t\r \u00a0 1.1.6.3\t\r \u00a0T3SS-\u00ad\u20102\t\r \u00a0translocons\t\r \u00a0Similar\t\r \u00a0to\t\r \u00a0T3SS-\u00ad\u20101,\t\r \u00a0a\t\r \u00a0protein\t\r \u00a0complex\t\r \u00a0encoded\t\r \u00a0within\t\r \u00a0SPI-\u00ad\u20102\t\r \u00a0forms\t\r \u00a0a\t\r \u00a0pore\t\r \u00a0in\t\r \u00a0the\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0membrane\t\r \u00a0to\t\r \u00a0enables\t\r \u00a0translocation\t\r \u00a0of\t\r \u00a0T3SS-\u00ad\u20102\t\r \u00a0effectors.\t\r \u00a0SseB,\t\r \u00a0SseC,\t\r \u00a0and\t\r \u00a0SseD\t\r \u00a0form\t\r \u00a0the\t\r \u00a0T3SS-\u00ad\u20102\t\r \u00a0translocon(230).\t\r \u00a0SseB\t\r \u00a0is\t\r \u00a0thought\t\r \u00a0to\t\r \u00a0form\t\r \u00a0the\t\r \u00a0sheath\t\r \u00a0appendage,\t\r \u00a0while\t\r \u00a0SseC\t\r \u00a0and\t\r \u00a0SseD\t\r \u00a0form\t\r \u00a0the\t\r \u00a0transmembrane\t\r \u00a0pore\t\r \u00a0at\t\r \u00a0the\t\r \u00a0tip\t\r \u00a0of\t\r \u00a0the\t\r \u00a0needle.\t\r \u00a0Interestingly,\t\r \u00a0a\t\r \u00a0SPI-\u00ad\u20102\t\r \u00a0encoded\t\r \u00a0complex\t\r \u00a0has\t\r \u00a0recently\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0regulate\t\r \u00a0a\t\r \u00a0transition\t\r \u00a0between\t\r \u00a0secretion\t\r \u00a0of\t\r \u00a0translocons\t\r \u00a0and\t\r \u00a0effectors\t\r \u00a0by\t\r \u00a0T3SS-\u00ad\u20102.\t\r \u00a0At\t\r \u00a0low\t\r \u00a0pH\t\r \u00a0within\t\r \u00a0the\t\r \u00a0SCV,\t\r \u00a0a\t\r \u00a0complex\t\r \u00a0of\t\r \u00a0SsaL,\t\r \u00a0SsaM\t\r \u00a0and\t\r \u00a0SpiC\t\r \u00a0forms\t\r \u00a0and\t\r \u00a0promotes\t\r \u00a0secretion\t\r \u00a0of\t\r \u00a0SseB,\t\r \u00a0SseC\t\r \u00a0and\t\r \u00a0SseD,\t\r \u00a0but\t\r \u00a0not\t\r \u00a0T3SS-\u00ad\u20102\t\r \u00a0effectors(231).\t\r \u00a0However,\t\r \u00a0when\t\r \u00a0the\t\r \u00a0translocon\t\r \u00a0forms\t\r \u00a0and\t\r \u00a0the\t\r \u00a0neutral\t\r \u00a0pH\t\r \u00a0of\t\r \u00a0the\t\r \u00a0host\t\r \u00a0cytosol\t\r \u00a0is\t\r \u00a0detected\t\r \u00a0by\t\r \u00a0the\t\r \u00a0bacteria,\t\r \u00a0the\t\r \u00a0SsaL,\t\r \u00a0SsaM,\t\r \u00a0SpiC\t\r \u00a0complex\t\r \u00a0disassociates\t\r \u00a0and\t\r \u00a0effector\t\r \u00a0translocation\t\r \u00a0is\t\r \u00a0enabled.\t\r \u00a0\t\r \u00a0 1.1.6.4\t\r \u00a0T3SS-\u00ad\u20102\t\r \u00a0effectors\t\r \u00a0More\t\r \u00a0than\t\r \u00a0twenty\t\r \u00a0effectors\t\r \u00a0are\t\r \u00a0known\t\r \u00a0to\t\r \u00a0be\t\r \u00a0translocated\t\r \u00a0by\t\r \u00a0Salmonella\t\r \u00a0T3SS-\u00ad\u20102.\t\r \u00a0While\t\r \u00a0the\t\r \u00a0functions\t\r \u00a0of\t\r \u00a0these\t\r \u00a0effectors\t\r \u00a0are\t\r \u00a0not\t\r \u00a0as\t\r \u00a0well\t\r \u00a0characterized\t\r \u00a0as\t\r \u00a0those\t\r \u00a0of\t\r \u00a0T3SS-\u00ad\u20101,\t\r \u00a0several\t\r \u00a0have\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0regulate\t\r \u00a0trafficking\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV\t\r \u00a0and\t\r \u00a0SIF\t\r \u00a0formation(Table\t\r \u00a01.2).\t\r \u00a0By\t\r \u00a0far\t\r \u00a0the\t\r \u00a0best\t\r \u00a0characterized\t\r \u00a0T3SS-\u00ad\u20102\t\r \u00a0effector\t\r \u00a0is\t\r \u00a0SifA.\t\r \u00a0 Salmonella\t\r \u00a0strains\t\r \u00a0lacking\t\r \u00a0SifA\t\r \u00a0are\t\r \u00a0heavily\t\r \u00a0attenuated\t\r \u00a0for\t\r \u00a0virulence,\t\r \u00a0do\t\r \u00a0not\t\r \u00a0form\t\r \u00a0SIFs\t\r \u00a0and\t\r \u00a0are\t\r \u00a0released\t\r \u00a0from\t\r \u00a0the\t\r \u00a0SCV\t\r \u00a0into\t\r \u00a0the\t\r \u00a0host\t\r \u00a0cytosol(94,\t\r \u00a0232,\t\r \u00a0233).\t\r \u00a0SifA\t\r \u00a0is\t\r \u00a0prenylated\t\r \u00a0 \t\r \u00a0\t\r \u00a0 27\t\r \u00a0 within\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0and\t\r \u00a0binds\t\r \u00a0Rab7\t\r \u00a0on\t\r \u00a0SCVs,\t\r \u00a0preventing\t\r \u00a0the\t\r \u00a0recruitment\t\r \u00a0of\t\r \u00a0dynein\t\r \u00a0to\t\r \u00a0regulate\t\r \u00a0SCV\t\r \u00a0positioning(68,\t\r \u00a0234).\t\r \u00a0SifA\t\r \u00a0also\t\r \u00a0binds\t\r \u00a0SifA\t\r \u00a0and\t\r \u00a0kinesin-\u00ad\u2010interacting\t\r \u00a0protein\t\r \u00a0(SKIP),\t\r \u00a0which\t\r \u00a0prevents\t\r \u00a0accumulation\t\r \u00a0of\t\r \u00a0kinesin\t\r \u00a0on\t\r \u00a0SCVs\t\r \u00a0and\t\r \u00a0is\t\r \u00a0required\t\r \u00a0for\t\r \u00a0SIF\t\r \u00a0formation(80,\t\r \u00a0235).\t\r \u00a0SifA\t\r \u00a0binding\t\r \u00a0to\t\r \u00a0SKIP\t\r \u00a0displaces\t\r \u00a0Rab9\t\r \u00a0from\t\r \u00a0SCVs,\t\r \u00a0which\t\r \u00a0is\t\r \u00a0required\t\r \u00a0for\t\r \u00a0LAMP1\t\r \u00a0recruitment\t\r \u00a0and\t\r \u00a0perinuclear\t\r \u00a0positioning(236).\t\r \u00a0Finally\t\r \u00a0SifA\t\r \u00a0also\t\r \u00a0interacts\t\r \u00a0with\t\r \u00a0RhoA,\t\r \u00a0which\t\r \u00a0is\t\r \u00a0dependent\t\r \u00a0on\t\r \u00a0its\t\r \u00a0WxxxE\t\r \u00a0motif\t\r \u00a0and\t\r \u00a0likely\t\r \u00a0activates\t\r \u00a0RhoA\t\r \u00a0to\t\r \u00a0induce\t\r \u00a0membrane\t\r \u00a0tubulation(237).\t\r \u00a0\t\r \u00a0Another\t\r \u00a0T3SS-\u00ad\u20102\t\r \u00a0effector,\t\r \u00a0SseJ,\t\r \u00a0also\t\r \u00a0binds\t\r \u00a0RhoA\t\r \u00a0and\t\r \u00a0can\t\r \u00a0induce\t\r \u00a0endosomal\t\r \u00a0tubulations\t\r \u00a0similar\t\r \u00a0to\t\r \u00a0SIFs\t\r \u00a0when\t\r \u00a0expressed\t\r \u00a0with\t\r \u00a0RhoA,B\t\r \u00a0or\t\r \u00a0C\t\r \u00a0suggesting\t\r \u00a0that\t\r \u00a0SifA\t\r \u00a0likely\t\r \u00a0mimics\t\r \u00a0Rho\t\r \u00a0GTPase\t\r \u00a0function\t\r \u00a0for\t\r \u00a0SIF\t\r \u00a0formation(237).\t\r \u00a0However\t\r \u00a0SseJ,\t\r \u00a0which\t\r \u00a0is\t\r \u00a0an\t\r \u00a0acyltransferase,\t\r \u00a0is\t\r \u00a0thought\t\r \u00a0to\t\r \u00a0negatively\t\r \u00a0regulate\t\r \u00a0SIF\t\r \u00a0formation\t\r \u00a0and\t\r \u00a0stability\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV,\t\r \u00a0as\t\r \u00a0expression\t\r \u00a0of\t\r \u00a0SseJ\t\r \u00a0inhibits\t\r \u00a0SIF\t\r \u00a0formation\t\r \u00a0by\t\r \u00a0wild\t\r \u00a0type\t\r \u00a0(WT)\t\r \u00a0bacteria\t\r \u00a0and\t\r \u00a0an\t\r \u00a0SseJ\t\r \u00a0and\t\r \u00a0SifA\t\r \u00a0double\t\r \u00a0mutant\t\r \u00a0restores\t\r \u00a0stability\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV(238,\t\r \u00a0239).\t\r \u00a0Similar\t\r \u00a0to\t\r \u00a0SseJ,\t\r \u00a0SopD2\t\r \u00a0is\t\r \u00a0also\t\r \u00a0thought\t\r \u00a0to\t\r \u00a0regulate\t\r \u00a0SIF\t\r \u00a0formation\t\r \u00a0and\t\r \u00a0to\t\r \u00a0oppose\t\r \u00a0SifA\t\r \u00a0function,\t\r \u00a0as\t\r \u00a0a\t\r \u00a0SopD2\t\r \u00a0and\t\r \u00a0SifA\t\r \u00a0double\t\r \u00a0mountain\t\r \u00a0retains\t\r \u00a0SCV\t\r \u00a0stability(240,\t\r \u00a0241).\t\r \u00a0\t\r \u00a0SseF\t\r \u00a0and\t\r \u00a0SseG\t\r \u00a0form\t\r \u00a0a\t\r \u00a0complex\t\r \u00a0and\t\r \u00a0associate\t\r \u00a0with\t\r \u00a0microtubules\t\r \u00a0to\t\r \u00a0promote\t\r \u00a0SIF\t\r \u00a0formation(242-\u00ad\u2010244).\t\r \u00a0In\t\r \u00a0addition,\t\r \u00a0SseF\t\r \u00a0has\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0prevent\t\r \u00a0dynein\t\r \u00a0recruitment,\t\r \u00a0while\t\r \u00a0SseG\t\r \u00a0has\t\r \u00a0been\t\r \u00a0reported\t\r \u00a0to\t\r \u00a0recruit\t\r \u00a0Golgi\t\r \u00a0membranes\t\r \u00a0to\t\r \u00a0the\t\r \u00a0SCV(245,\t\r \u00a0246).\t\r \u00a0PipB2\t\r \u00a0also\t\r \u00a0promotes\t\r \u00a0SIF\t\r \u00a0formation\t\r \u00a0by\t\r \u00a0binding\t\r \u00a0kinesin-\u00ad\u20101\t\r \u00a0and\t\r \u00a0promoting\t\r \u00a0extension\t\r \u00a0of\t\r \u00a0SIF\t\r \u00a0away\t\r \u00a0from\t\r \u00a0the\t\r \u00a0SCV\t\r \u00a0along\t\r \u00a0microtubules(247,\t\r \u00a0248).\t\r \u00a0Finally\t\r \u00a0SpiC\t\r \u00a0is\t\r \u00a0also\t\r \u00a0thought\t\r \u00a0to\t\r \u00a0regulate\t\r \u00a0SCV\t\r \u00a0trafficking\t\r \u00a0though\t\r \u00a0interactions\t\r \u00a0with\t\r \u00a0Target\t\r \u00a0for\t\r \u00a0Salmonella\t\r \u00a0secreted\t\r \u00a0protein\t\r \u00a0C\t\r \u00a0(TassC)\t\r \u00a0and\t\r \u00a0Protein\t\r \u00a0Hook\t\r \u00a0homolog\t\r \u00a03\t\r \u00a0(Hook3)(249-\u00ad\u2010251).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 28\t\r \u00a0 Several\t\r \u00a0T3SS-\u00ad\u20102\t\r \u00a0effectors\t\r \u00a0also\t\r \u00a0regulate\t\r \u00a0the\t\r \u00a0formation\t\r \u00a0of\t\r \u00a0VAP\t\r \u00a0around\t\r \u00a0the\t\r \u00a0SCV.\t\r \u00a0SpvB\t\r \u00a0has\t\r \u00a0a\t\r \u00a0profound\t\r \u00a0negative\t\r \u00a0affect\t\r \u00a0on\t\r \u00a0VAP\t\r \u00a0formation\t\r \u00a0by\t\r \u00a0ribosylating\t\r \u00a0actin\t\r \u00a0and\t\r \u00a0preventing\t\r \u00a0polymerization(252-\u00ad\u2010254).\t\r \u00a0SseI\t\r \u00a0and\t\r \u00a0SspH2\t\r \u00a0interact\t\r \u00a0with\t\r \u00a0filamin,\t\r \u00a0and\t\r \u00a0SspH2\t\r \u00a0interacts\t\r \u00a0with\t\r \u00a0profilin\t\r \u00a0to\t\r \u00a0inhibit\t\r \u00a0the\t\r \u00a0rate\t\r \u00a0of\t\r \u00a0actin\t\r \u00a0polymerization(255).\t\r \u00a0SteC\t\r \u00a0is\t\r \u00a0a\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a0and\t\r \u00a0has\t\r \u00a0also\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0cause\t\r \u00a0rearrangements\t\r \u00a0of\t\r \u00a0the\t\r \u00a0actin\t\r \u00a0cytoskeleton\t\r \u00a0through\t\r \u00a0an\t\r \u00a0unknown\t\r \u00a0mechanism(256).\t\r \u00a0Additional\t\r \u00a0activities\t\r \u00a0of\t\r \u00a0T3SS-\u00ad\u20102\t\r \u00a0effectors\t\r \u00a0include\t\r \u00a0deubiquitylase\t\r \u00a0activity\t\r \u00a0of\t\r \u00a0SseL,\t\r \u00a0which,\t\r \u00a0similar\t\r \u00a0to\t\r \u00a0AvrA,\t\r \u00a0prevents\t\r \u00a0ubiquitylation\t\r \u00a0and\t\r \u00a0degradation\t\r \u00a0of\t\r \u00a0I-\u00ad\u2010\u03baB\u03b1\t\r \u00a0to\t\r \u00a0reduce\t\r \u00a0host\t\r \u00a0inflammation,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0SpvC,\t\r \u00a0which\t\r \u00a0is\t\r \u00a0a\t\r \u00a0phosphothreonine\t\r \u00a0lyase\t\r \u00a0and\t\r \u00a0downregulates\t\r \u00a0ERK1\/2\t\r \u00a0activity(257,\t\r \u00a0258).\t\r \u00a0SseJ\t\r \u00a0has\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0esterify\t\r \u00a0cholesterol\t\r \u00a0and\t\r \u00a0reduce\t\r \u00a0cholesterol\t\r \u00a0levels\t\r \u00a0in\t\r \u00a0host\t\r \u00a0membranes,\t\r \u00a0and\t\r \u00a0SseI\t\r \u00a0interacts\t\r \u00a0with\t\r \u00a0Ras\t\r \u00a0GTPase-\u00ad\u2010activating-\u00ad\u2010like\t\r \u00a0protein\t\r \u00a0IQGAP1\t\r \u00a0(IQGAP1)\t\r \u00a0and\t\r \u00a0Thyroid\t\r \u00a0receptor-\u00ad\u2010interacting\t\r \u00a0protein\t\r \u00a06\t\r \u00a0(TRIP6)\t\r \u00a0which\t\r \u00a0regulates\t\r \u00a0phagocyte\t\r \u00a0motility\t\r \u00a0and\t\r \u00a0has\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0be\t\r \u00a0involved\t\r \u00a0in\t\r \u00a0systemic\t\r \u00a0spread\t\r \u00a0of\t\r \u00a0infection(259).\t\r \u00a0\t\r \u00a0\t\r \u00a0\t\r \u00a0\t\r \u00a0\t\r \u00a0\t\r \u00a0\t\r \u00a0\t\r \u00a0\t\r \u00a0 \t\r \u00a0\t\r \u00a0 29\t\r \u00a0 Table\t\r \u00a01.2\t\r \u00a0Functions\t\r \u00a0and\t\r \u00a0host\t\r \u00a0targets\t\r \u00a0of\t\r \u00a0T3SS-\u00ad\u20102\t\r \u00a0effectors\t\r \u00a0 Effector\t\r \u00a0 Cellular\t\r \u00a0activity\t\r \u00a0 Host-\u00ad\u2010target\t\r \u00a0 Reference\t\r \u00a0GogB\t\r \u00a0 Unknown\t\r \u00a0 unknown\t\r \u00a0 (260)\t\r \u00a0PipB\t\r \u00a0 Unknown.\t\r \u00a0Localizes\t\r \u00a0to\t\r \u00a0SCVs\t\r \u00a0and\t\r \u00a0SIFs.\t\r \u00a0 unknown\t\r \u00a0 (72)\t\r \u00a0PipB2\t\r \u00a0 Binds\t\r \u00a0kinesin-\u00ad\u20101,\t\r \u00a0causes\t\r \u00a0extension\t\r \u00a0of\t\r \u00a0SIFs\t\r \u00a0on\t\r \u00a0microtubules.\t\r \u00a0Localizes\t\r \u00a0to\t\r \u00a0SCVs,\t\r \u00a0SIFs,\t\r \u00a0and\t\r \u00a0vesicles\t\r \u00a0at\t\r \u00a0cell\t\r \u00a0periphery.\t\r \u00a0 Kinesin-\u00ad\u20101\t\r \u00a0 (72,\t\r \u00a0247,\t\r \u00a0248)\t\r \u00a0 SifA\t\r \u00a0 Induces\t\r \u00a0SIF\t\r \u00a0formation\t\r \u00a0and\t\r \u00a0SCV\t\r \u00a0stability.\t\r \u00a0Binds\t\r \u00a0Rab7\t\r \u00a0to\t\r \u00a0prevent\t\r \u00a0RILP\t\r \u00a0recruitment\t\r \u00a0to\t\r \u00a0SCV.\t\r \u00a0Binds\t\r \u00a0SKIP\t\r \u00a0to\t\r \u00a0restrict\t\r \u00a0kinesin\t\r \u00a0activity\t\r \u00a0on\t\r \u00a0SCV\t\r \u00a0and\t\r \u00a0displace\t\r \u00a0Rab9\t\r \u00a0from\t\r \u00a0SCVs.\t\r \u00a0Binds\t\r \u00a0RhoA,\t\r \u00a0likely\t\r \u00a0acting\t\r \u00a0as\t\r \u00a0a\t\r \u00a0GEF\t\r \u00a0to\t\r \u00a0promote\t\r \u00a0SIF\t\r \u00a0formation.\t\r \u00a0 Rab7,\t\r \u00a0SKIP,\t\r \u00a0RhoA\t\r \u00a0 (68,\t\r \u00a080,\t\r \u00a094,\t\r \u00a0232,\t\r \u00a0235-\u00ad\u2010237)\t\r \u00a0 SifB\t\r \u00a0 Unknown.\t\r \u00a0Localizes\t\r \u00a0to\t\r \u00a0SCVs\t\r \u00a0and\t\r \u00a0SIFs.\t\r \u00a0 unknown\t\r \u00a0 (261)\t\r \u00a0SopD2\t\r \u00a0 Contributes\t\r \u00a0to\t\r \u00a0SIF\t\r \u00a0formation\t\r \u00a0and\t\r \u00a0SCV\t\r \u00a0stability\t\r \u00a0by\t\r \u00a0opposing\t\r \u00a0actions\t\r \u00a0of\t\r \u00a0SifA.\t\r \u00a0Localizes\t\r \u00a0to\t\r \u00a0SCVs\t\r \u00a0and\t\r \u00a0SIFs.\t\r \u00a0 unknown\t\r \u00a0 (240,\t\r \u00a0241,\t\r \u00a0262)\t\r \u00a0 SpiC\t\r \u00a0 Regulates\t\r \u00a0SCV\t\r \u00a0trafficking,\t\r \u00a0likely\t\r \u00a0by\t\r \u00a0binding\t\r \u00a0TassC\t\r \u00a0and\t\r \u00a0Hook3.\t\r \u00a0 TassC,\t\r \u00a0Hook3\t\r \u00a0 (249-\u00ad\u2010251)\t\r \u00a0 SpvB\t\r \u00a0 ADP-\u00ad\u2010ribosyltransferase\t\r \u00a0that\t\r \u00a0targets\t\r \u00a0actin\t\r \u00a0to\t\r \u00a0prevent\t\r \u00a0formation\t\r \u00a0of\t\r \u00a0F-\u00ad\u2010actin.\t\r \u00a0Downregulates\t\r \u00a0VAP\t\r \u00a0and\t\r \u00a0SIF\t\r \u00a0formation.\t\r \u00a0 actin\t\r \u00a0 (75,\t\r \u00a0252-\u00ad\u2010255)\t\r \u00a0 SpvC\t\r \u00a0 Reduces\t\r \u00a0cytokine\t\r \u00a0release\t\r \u00a0from\t\r \u00a0infected\t\r \u00a0cells\t\r \u00a0and\t\r \u00a0has\t\r \u00a0phosphothreonine\t\r \u00a0lyase\t\r \u00a0activity.\t\r \u00a0 ERK1\/2\t\r \u00a0 (258,\t\r \u00a0263)\t\r \u00a0 SseF\t\r \u00a0 Contributes\t\r \u00a0to\t\r \u00a0SIF\t\r \u00a0formation,\t\r \u00a0microtubule\t\r \u00a0bundling,\t\r \u00a0and\t\r \u00a0positioning\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV.\t\r \u00a0Binds\t\r \u00a0SseG.\t\r \u00a0Prevents\t\r \u00a0recruitment\t\r \u00a0of\t\r \u00a0dynein\t\r \u00a0to\t\r \u00a0SCV.\t\r \u00a0 unknown\t\r \u00a0\t\r \u00a0 (242-\u00ad\u2010245)\t\r \u00a0 SseG\t\r \u00a0 Contributes\t\r \u00a0to\t\r \u00a0SIF\t\r \u00a0formation,\t\r \u00a0microtubule\t\r \u00a0bundling,\t\r \u00a0and\t\r \u00a0positioning\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV.\t\r \u00a0Binds\t\r \u00a0SseF.\t\r \u00a0Targets\t\r \u00a0SCV\t\r \u00a0to\t\r \u00a0the\t\r \u00a0Golgi.\t\r \u00a0 unknown\t\r \u00a0 (242-\u00ad\u2010244,\t\r \u00a0246)\t\r \u00a0 \t\r \u00a0\t\r \u00a0 30\t\r \u00a0 Effector\t\r \u00a0 Cellular\t\r \u00a0activity\t\r \u00a0 Host-\u00ad\u2010target\t\r \u00a0 Reference\t\r \u00a0SseI\/SrfH\t\r \u00a0 Binds\t\r \u00a0filamin\t\r \u00a0and\t\r \u00a0localizes\t\r \u00a0to\t\r \u00a0actin\t\r \u00a0cytoskeleton.\t\r \u00a0Interacts\t\r \u00a0with\t\r \u00a0IQGAP1\t\r \u00a0and\t\r \u00a0TRIP6\t\r \u00a0to\t\r \u00a0regulate\t\r \u00a0motility\t\r \u00a0of\t\r \u00a0macrophages\t\r \u00a0and\t\r \u00a0dendritic\t\r \u00a0cells.\t\r \u00a0 filamin,\t\r \u00a0IQGAP1,\t\r \u00a0TRIP6\t\r \u00a0 (255,\t\r \u00a0264,\t\r \u00a0265)\t\r \u00a0 SseJ\t\r \u00a0 Negatively\t\r \u00a0regulates\t\r \u00a0SIF\t\r \u00a0formation\t\r \u00a0and\t\r \u00a0SCV\t\r \u00a0stability.\t\r \u00a0Acyltransferase\t\r \u00a0activity.\t\r \u00a0Esterifies\t\r \u00a0cholesterol.\t\r \u00a0 RhoA,\t\r \u00a0cholesterol\t\r \u00a0 (237-\u00ad\u2010239,\t\r \u00a0259,\t\r \u00a0261,\t\r \u00a0266)\t\r \u00a0 SseK1\t\r \u00a0 unknown\t\r \u00a0 unknown\t\r \u00a0 (267)\t\r \u00a0SseK2\t\r \u00a0 unknown\t\r \u00a0 unknown\t\r \u00a0 (267)\t\r \u00a0SseL\t\r \u00a0 Deubiquitinase,\t\r \u00a0decreases\t\r \u00a0NF-\u00ad\u2010\u03baB\t\r \u00a0activation\t\r \u00a0reducing\t\r \u00a0inflammatory\t\r \u00a0responses\t\r \u00a0in\t\r \u00a0host.\t\r \u00a0 ubiquitin,\t\r \u00a0I-\u00ad\u2010\u03baB\u03b1\t\r \u00a0 (268)\t\r \u00a0(257)\t\r \u00a0 SspH2\t\r \u00a0 Colocalizes\t\r \u00a0with\t\r \u00a0actin\t\r \u00a0cytoskeleton\t\r \u00a0and\t\r \u00a0reduces\t\r \u00a0rate\t\r \u00a0of\t\r \u00a0actin\t\r \u00a0polymerization.\t\r \u00a0Interacts\t\r \u00a0with\t\r \u00a0filamin\t\r \u00a0and\t\r \u00a0profilin.\t\r \u00a0 filamin,\t\r \u00a0profilin\t\r \u00a0 (255)\t\r \u00a0 SteA\t\r \u00a0 unknown\t\r \u00a0 unknown\t\r \u00a0 (269)\t\r \u00a0SteB\t\r \u00a0 unknown\t\r \u00a0 unknown\t\r \u00a0 (269)\t\r \u00a0SteC\t\r \u00a0 Protein\t\r \u00a0kinase\t\r \u00a0activity\t\r \u00a0causes\t\r \u00a0F-\u00ad\u2010actin\t\r \u00a0rearrangements.\t\r \u00a0 unknown\t\r \u00a0 (256,\t\r \u00a0269)\t\r \u00a0SlrP\t\r \u00a0 E3\t\r \u00a0ubiquitin\t\r \u00a0ligase.\t\r \u00a0Targets\t\r \u00a0thioredoxin\t\r \u00a0for\t\r \u00a0degradation,\t\r \u00a0promoting\t\r \u00a0cell\t\r \u00a0death.\t\r \u00a0Also\t\r \u00a0targets\t\r \u00a0ER\t\r \u00a0chaperone\t\r \u00a0ERdj3.\t\r \u00a0 thioredoxin,\t\r \u00a0ERdj3\t\r \u00a0 (180,\t\r \u00a0181)\t\r \u00a0 SspH1\t\r \u00a0 Inhibits\t\r \u00a0NF-\u00ad\u2010\u03baB\t\r \u00a0dependent\t\r \u00a0gen\t\r \u00a0expression.\t\r \u00a0Interacts\t\r \u00a0with\t\r \u00a0PKN1\t\r \u00a0and\t\r \u00a0had\t\r \u00a0E3\t\r \u00a0ubiquitin\t\r \u00a0ligase\t\r \u00a0activity.\t\r \u00a0 PKN1\t\r \u00a0 (182-\u00ad\u2010184)\t\r \u00a0 \t\r \u00a0 1.1.6.5\t\r \u00a0T3SS-\u00ad\u20102\t\r \u00a0chaperones\t\r \u00a0Several\t\r \u00a0chaperones\t\r \u00a0have\t\r \u00a0also\t\r \u00a0been\t\r \u00a0found\t\r \u00a0to\t\r \u00a0regulate\t\r \u00a0secretion\t\r \u00a0of\t\r \u00a0translocons\t\r \u00a0and\t\r \u00a0effectors\t\r \u00a0through\t\r \u00a0T3SS-\u00ad\u20102.\t\r \u00a0These\t\r \u00a0include\t\r \u00a0SseA,\t\r \u00a0which\t\r \u00a0is\t\r \u00a0a\t\r \u00a0reported\t\r \u00a0chaperone\t\r \u00a0for\t\r \u00a0the\t\r \u00a0translocons\t\r \u00a0SseB\t\r \u00a0and\t\r \u00a0SseD(270-\u00ad\u2010272).\t\r \u00a0However,\t\r \u00a0more\t\r \u00a0recently\t\r \u00a0SsaE\t\r \u00a0has\t\r \u00a0also\t\r \u00a0 \t\r \u00a0\t\r \u00a0 31\t\r \u00a0 been\t\r \u00a0proposed\t\r \u00a0to\t\r \u00a0act\t\r \u00a0as\t\r \u00a0a\t\r \u00a0chaperone\t\r \u00a0for\t\r \u00a0SseB(273).\t\r \u00a0Furthermore,\t\r \u00a0SscB\t\r \u00a0is\t\r \u00a0a\t\r \u00a0chaperone\t\r \u00a0for\t\r \u00a0SseF,\t\r \u00a0and\t\r \u00a0SrcA\t\r \u00a0is\t\r \u00a0a\t\r \u00a0chaperone\t\r \u00a0for\t\r \u00a0both\t\r \u00a0SseL\t\r \u00a0and\t\r \u00a0PipB2(274).\t\r \u00a0\t\r \u00a0 1.2\t\r \u00a0Mass\t\r \u00a0spectrometry-\u00ad\u2010based\t\r \u00a0proteomics\t\r \u00a0 1.2.1\t\r \u00a0General\t\r \u00a0proteomics\t\r \u00a0Originally\t\r \u00a0in\t\r \u00a01994,\t\r \u00a0the\t\r \u00a0proteome\t\r \u00a0was\t\r \u00a0defined\t\r \u00a0as\t\r \u00a0the\t\r \u00a0protein\t\r \u00a0complement\t\r \u00a0of\t\r \u00a0a\t\r \u00a0genome,\t\r \u00a0and\t\r \u00a0the\t\r \u00a0process\t\r \u00a0of\t\r \u00a0studying\t\r \u00a0a\t\r \u00a0proteome\t\r \u00a0became\t\r \u00a0known\t\r \u00a0as\t\r \u00a0proteomics(275).\t\r \u00a0Today,\t\r \u00a0a\t\r \u00a0proteome\t\r \u00a0is\t\r \u00a0defined\t\r \u00a0as\t\r \u00a0the\t\r \u00a0protein\t\r \u00a0complement\t\r \u00a0of\t\r \u00a0a\t\r \u00a0genome,\t\r \u00a0cell,\t\r \u00a0tissue\t\r \u00a0or\t\r \u00a0organism\t\r \u00a0at\t\r \u00a0a\t\r \u00a0given\t\r \u00a0time\t\r \u00a0and\t\r \u00a0under\t\r \u00a0a\t\r \u00a0defined\t\r \u00a0set\t\r \u00a0of\t\r \u00a0conditions(276,\t\r \u00a0277)).\t\r \u00a0Thus,\t\r \u00a0in\t\r \u00a0contrast\t\r \u00a0to\t\r \u00a0genomes,\t\r \u00a0which\t\r \u00a0are\t\r \u00a0relatively\t\r \u00a0static,\t\r \u00a0proteomes\t\r \u00a0represent\t\r \u00a0the\t\r \u00a0complement\t\r \u00a0of\t\r \u00a0expressed\t\r \u00a0genes\t\r \u00a0in\t\r \u00a0space\t\r \u00a0and\t\r \u00a0time.\t\r \u00a0Due\t\r \u00a0to\t\r \u00a0this\t\r \u00a0dynamic\t\r \u00a0nature,\t\r \u00a0the\t\r \u00a0fact\t\r \u00a0that\t\r \u00a0genes\t\r \u00a0can\t\r \u00a0encode\t\r \u00a0multiple\t\r \u00a0proteins\t\r \u00a0by\t\r \u00a0alternative\t\r \u00a0splicing,\t\r \u00a0and\t\r \u00a0the\t\r \u00a0profound\t\r \u00a0degree\t\r \u00a0of\t\r \u00a0regulation\t\r \u00a0(proteolysis,\t\r \u00a0glycolysis,\t\r \u00a0ubiquitylation,\t\r \u00a0phosphorylation,\t\r \u00a0etc.)\t\r \u00a0observed\t\r \u00a0at\t\r \u00a0the\t\r \u00a0protein\t\r \u00a0level,\t\r \u00a0proteomes\t\r \u00a0offer\t\r \u00a0a\t\r \u00a0great\t\r \u00a0deal\t\r \u00a0of\t\r \u00a0information\t\r \u00a0into\t\r \u00a0biological\t\r \u00a0activities.\t\r \u00a0Originally,\t\r \u00a0Edman\t\r \u00a0sequencing\t\r \u00a0was\t\r \u00a0the\t\r \u00a0most\t\r \u00a0common\t\r \u00a0means\t\r \u00a0of\t\r \u00a0protein\t\r \u00a0identification\t\r \u00a0for\t\r \u00a0proteomics\t\r \u00a0approaches(278,\t\r \u00a0279).\t\r \u00a0While\t\r \u00a0this\t\r \u00a0technique\t\r \u00a0is\t\r \u00a0reliable\t\r \u00a0and\t\r \u00a0can\t\r \u00a0be\t\r \u00a0automated,\t\r \u00a0it\t\r \u00a0is\t\r \u00a0slow,\t\r \u00a0suffers\t\r \u00a0from\t\r \u00a0low\t\r \u00a0sensitivity,\t\r \u00a0and\t\r \u00a0the\t\r \u00a0databases\t\r \u00a0used\t\r \u00a0were\t\r \u00a0originally\t\r \u00a0quite\t\r \u00a0limited.\t\r \u00a0However,\t\r \u00a0following\t\r \u00a0sequencing\t\r \u00a0of\t\r \u00a0genomes\t\r \u00a0such\t\r \u00a0as\t\r \u00a0yeast\t\r \u00a0in\t\r \u00a01996\t\r \u00a0and\t\r \u00a0humans\t\r \u00a0in\t\r \u00a02001,\t\r \u00a0our\t\r \u00a0ability\t\r \u00a0to\t\r \u00a0identify\t\r \u00a0proteins\t\r \u00a0relied\t\r \u00a0only\t\r \u00a0on\t\r \u00a0our\t\r \u00a0capacity\t\r \u00a0to\t\r \u00a0extract\t\r \u00a0sequence\t\r \u00a0information\t\r \u00a0and\t\r \u00a0correlate\t\r \u00a0it\t\r \u00a0with\t\r \u00a0the\t\r \u00a0emerging\t\r \u00a0sequence\t\r \u00a0databases(280-\u00ad\u2010282).\t\r \u00a0Originally,\t\r \u00a0peptide\t\r \u00a0mass\t\r \u00a0fingerprinting\t\r \u00a0was\t\r \u00a0developed,\t\r \u00a0where\t\r \u00a0mass\t\r \u00a0spectrometry\t\r \u00a0is\t\r \u00a0used\t\r \u00a0to\t\r \u00a0determine\t\r \u00a0the\t\r \u00a0masses\t\r \u00a0of\t\r \u00a0ionized\t\r \u00a0 \t\r \u00a0\t\r \u00a0 32\t\r \u00a0 peptides,\t\r \u00a0which\t\r \u00a0are\t\r \u00a0then\t\r \u00a0matched\t\r \u00a0to\t\r \u00a0sequence\t\r \u00a0databases\t\r \u00a0for\t\r \u00a0protein\t\r \u00a0identification(283,\t\r \u00a0284).\t\r \u00a0This\t\r \u00a0technique\t\r \u00a0was\t\r \u00a0often\t\r \u00a0used\t\r \u00a0in\t\r \u00a0combination\t\r \u00a0with\t\r \u00a0two-\u00ad\u2010dimensional\t\r \u00a0gel\t\r \u00a0electrophoresis\t\r \u00a0(2DE)(285,\t\r \u00a0286).\t\r \u00a0However,\t\r \u00a0despite\t\r \u00a0its\t\r \u00a0outstanding\t\r \u00a0resolving\t\r \u00a0power,\t\r \u00a02DE\t\r \u00a0it\t\r \u00a0is\t\r \u00a0generally\t\r \u00a0plagued\t\r \u00a0with\t\r \u00a0low\t\r \u00a0reproducibility\t\r \u00a0and\t\r \u00a0other\t\r \u00a0technical\t\r \u00a0problems,\t\r \u00a0and\t\r \u00a0peptide\t\r \u00a0mass\t\r \u00a0fingerprinting\t\r \u00a0is\t\r \u00a0not\t\r \u00a0sensitive\t\r \u00a0enough\t\r \u00a0for\t\r \u00a0whole\t\r \u00a0proteome\t\r \u00a0analysis(287,\t\r \u00a0288).\t\r \u00a0In\t\r \u00a0addition,\t\r \u00a0array-\u00ad\u2010based\t\r \u00a0techniques\t\r \u00a0have\t\r \u00a0also\t\r \u00a0been\t\r \u00a0employed\t\r \u00a0for\t\r \u00a0proteomics(289,\t\r \u00a0290).\t\r \u00a0However,\t\r \u00a0problems\t\r \u00a0including\t\r \u00a0cross-\u00ad\u2010reactivity\t\r \u00a0and\t\r \u00a0interference\t\r \u00a0by\t\r \u00a0affinity\t\r \u00a0tags\t\r \u00a0have\t\r \u00a0limited\t\r \u00a0their\t\r \u00a0applicability\t\r \u00a0as\t\r \u00a0well(291).\t\r \u00a0Today,\t\r \u00a0most\t\r \u00a0proteomics\t\r \u00a0applications\t\r \u00a0employ\t\r \u00a0methods\t\r \u00a0in\t\r \u00a0liquid\t\r \u00a0chromatography-\u00ad\u2010tandem\t\r \u00a0mass\t\r \u00a0spectrometry\t\r \u00a0(LC-\u00ad\u2010MSn)\t\r \u00a0for\t\r \u00a0protein\t\r \u00a0identification.\t\r \u00a0This\t\r \u00a0includes\t\r \u00a0top-\u00ad\u2010down\t\r \u00a0proteomics,\t\r \u00a0where\t\r \u00a0whole\t\r \u00a0proteins\t\r \u00a0are\t\r \u00a0ionized\t\r \u00a0and\t\r \u00a0characterized(292).\t\r \u00a0However,\t\r \u00a0bottom-\u00ad\u2010up\t\r \u00a0or\t\r \u00a0shot-\u00ad\u2010gun\t\r \u00a0proteomics\t\r \u00a0is\t\r \u00a0by\t\r \u00a0far\t\r \u00a0the\t\r \u00a0most\t\r \u00a0common.\t\r \u00a0In\t\r \u00a0this\t\r \u00a0case\t\r \u00a0a\t\r \u00a0protein\t\r \u00a0sample\t\r \u00a0is\t\r \u00a0enzymatically\t\r \u00a0digested,\t\r \u00a0and\t\r \u00a0the\t\r \u00a0resulting\t\r \u00a0peptides\t\r \u00a0are\t\r \u00a0resolved\t\r \u00a0by\t\r \u00a0liquid\t\r \u00a0chromatography,\t\r \u00a0and\t\r \u00a0fragmented\t\r \u00a0and\t\r \u00a0analyzed\t\r \u00a0within\t\r \u00a0a\t\r \u00a0mass\t\r \u00a0spectrometer(293,\t\r \u00a0294).\t\r \u00a0This\t\r \u00a0technique\t\r \u00a0has\t\r \u00a0been\t\r \u00a0used\t\r \u00a0to\t\r \u00a0characterize\t\r \u00a0systems\t\r \u00a0ranging\t\r \u00a0from\t\r \u00a0protein\t\r \u00a0complexes,\t\r \u00a0protein\t\r \u00a0localization,\t\r \u00a0cell\t\r \u00a0signaling,\t\r \u00a0and\t\r \u00a0protein\t\r \u00a0expression(295-\u00ad\u2010298).\t\r \u00a0\t\r \u00a0 1.2.2\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0In\t\r \u00a0a\t\r \u00a0typical\t\r \u00a0bottom-\u00ad\u2010up\t\r \u00a0proteomics\t\r \u00a0experiment,\t\r \u00a0proteins\t\r \u00a0within\t\r \u00a0a\t\r \u00a0biological\t\r \u00a0sample\t\r \u00a0are\t\r \u00a0enzymatically\t\r \u00a0digested,\t\r \u00a0most\t\r \u00a0often\t\r \u00a0with\t\r \u00a0trypsin\t\r \u00a0(Figure\t\r \u00a01.3)(294).\t\r \u00a0These\t\r \u00a0peptides\t\r \u00a0are\t\r \u00a0then\t\r \u00a0resolved\t\r \u00a0by\t\r \u00a0nano-\u00ad\u2010scale\t\r \u00a0reversed-\u00ad\u2010phase\t\r \u00a0high-\u00ad\u2010performance\t\r \u00a0liquid\t\r \u00a0 \t\r \u00a0\t\r \u00a0 33\t\r \u00a0 chromatography\t\r \u00a0(HPLC).\t\r \u00a0Peptides\t\r \u00a0eluting\t\r \u00a0from\t\r \u00a0the\t\r \u00a0chromatography\t\r \u00a0column\t\r \u00a0are\t\r \u00a0ionized\t\r \u00a0by\t\r \u00a0electrospray\t\r \u00a0ionization\t\r \u00a0(ESI),\t\r \u00a0which\t\r \u00a0couples\t\r \u00a0the\t\r \u00a0LC\t\r \u00a0system\t\r \u00a0to\t\r \u00a0one\t\r \u00a0of\t\r \u00a0several\t\r \u00a0types\t\r \u00a0of\t\r \u00a0mass\t\r \u00a0spectrometers.\t\r \u00a0Within\t\r \u00a0the\t\r \u00a0mass\t\r \u00a0spectrometer,\t\r \u00a0an\t\r \u00a0initial\t\r \u00a0parent\t\r \u00a0ion\t\r \u00a0scan\t\r \u00a0determines\t\r \u00a0the\t\r \u00a0mass\t\r \u00a0to\t\r \u00a0charge\t\r \u00a0ratio\t\r \u00a0(m\/z)\t\r \u00a0of\t\r \u00a0the\t\r \u00a0isolated\t\r \u00a0peptides,\t\r \u00a0several\t\r \u00a0of\t\r \u00a0which\t\r \u00a0are\t\r \u00a0retained\t\r \u00a0for\t\r \u00a0fragmentation\t\r \u00a0and\t\r \u00a0an\t\r \u00a0additional\t\r \u00a0round\t\r \u00a0or\t\r \u00a0rounds\t\r \u00a0of\t\r \u00a0MS.\t\r \u00a0Here,\t\r \u00a0fragmentation\t\r \u00a0is\t\r \u00a0most\t\r \u00a0often\t\r \u00a0achieved\t\r \u00a0at\t\r \u00a0peptide\t\r \u00a0bonds\t\r \u00a0by\t\r \u00a0collision-\u00ad\u2010induced\t\r \u00a0dissociation\t\r \u00a0(CID),\t\r \u00a0and\t\r \u00a0the\t\r \u00a0m\/z\t\r \u00a0of\t\r \u00a0the\t\r \u00a0fragments\t\r \u00a0is\t\r \u00a0detected\t\r \u00a0and\t\r \u00a0can\t\r \u00a0be\t\r \u00a0used\t\r \u00a0to\t\r \u00a0search\t\r \u00a0spectral\t\r \u00a0libraries\t\r \u00a0for\t\r \u00a0protein\t\r \u00a0identification.\t\r \u00a0In\t\r \u00a0most\t\r \u00a0cases,\t\r \u00a0one\t\r \u00a0round\t\r \u00a0of\t\r \u00a0CID\t\r \u00a0is\t\r \u00a0employed.\t\r \u00a0However,\t\r \u00a0in\t\r \u00a0cases\t\r \u00a0where\t\r \u00a0satisfactory\t\r \u00a0fragmentation\t\r \u00a0is\t\r \u00a0not\t\r \u00a0achieved,\t\r \u00a0the\t\r \u00a0fragments\t\r \u00a0can\t\r \u00a0be\t\r \u00a0re-\u00ad\u2010activated\t\r \u00a0by\t\r \u00a0additional\t\r \u00a0rounds\t\r \u00a0of\t\r \u00a0CID\t\r \u00a0to\t\r \u00a0generate\t\r \u00a0higher\t\r \u00a0order\t\r \u00a0MSn\t\r \u00a0spectra.\t\r \u00a0\t\r \u00a0Given\t\r \u00a0the\t\r \u00a0high\t\r \u00a0dynamic\t\r \u00a0range\t\r \u00a0of\t\r \u00a0biological\t\r \u00a0samples,\t\r \u00a0fractionation\t\r \u00a0and\t\r \u00a0enrichment\t\r \u00a0techniques\t\r \u00a0prior\t\r \u00a0to\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0are\t\r \u00a0often\t\r \u00a0required\t\r \u00a0for\t\r \u00a0complete\t\r \u00a0or\t\r \u00a0near\t\r \u00a0complete\t\r \u00a0proteome\t\r \u00a0coverage.\t\r \u00a0Furthermore,\t\r \u00a0MS\t\r \u00a0data\t\r \u00a0can\t\r \u00a0also\t\r \u00a0be\t\r \u00a0used\t\r \u00a0to\t\r \u00a0quantify\t\r \u00a0protein\t\r \u00a0abundance\t\r \u00a0and\t\r \u00a0to\t\r \u00a0localize\t\r \u00a0and\t\r \u00a0identify\t\r \u00a0post-\u00ad\u2010translational\t\r \u00a0modifications\t\r \u00a0(PTMs)\t\r \u00a0within\t\r \u00a0the\t\r \u00a0analyzed\t\r \u00a0peptides.\t\r \u00a0The\t\r \u00a0following\t\r \u00a0sections\t\r \u00a0contain\t\r \u00a0additional\t\r \u00a0descriptions\t\r \u00a0of\t\r \u00a0protein\t\r \u00a0identification\t\r \u00a0by\t\r \u00a0LC-\u00ad\u2010MSn,\t\r \u00a0quantitation\t\r \u00a0and\t\r \u00a0pre-\u00ad\u2010fractionation\t\r \u00a0techniques,\t\r \u00a0and\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0PTMs.\t\r \u00a0 \t\r \u00a0\t\r \u00a0 34\t\r \u00a0 Figure\t\r \u00a01.3\t\r \u00a0Schematic\t\r \u00a0of\t\r \u00a0a\t\r \u00a0bottom-\u00ad\u2010up\t\r \u00a0proteomics\t\r \u00a0workflow\t\r \u00a0 \t\r \u00a0A\t\r \u00a0basic\t\r \u00a0workflow\t\r \u00a0consisting\t\r \u00a0of\t\r \u00a0protein\t\r \u00a0sample\t\r \u00a0preparation,\t\r \u00a0proteolysis,\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0and\t\r \u00a0peptide\t\r \u00a0and\t\r \u00a0protein\t\r \u00a0identification\t\r \u00a0for\t\r \u00a0various\t\r \u00a0applications\t\r \u00a0is\t\r \u00a0shown,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0incorporating\t\r \u00a0quantitation\t\r \u00a0and\t\r \u00a0pre-\u00ad\u2010fractionation\t\r \u00a0methods.\t\r \u00a0\t\r \u00a0 1.2.3\t\r \u00a0LTQ-\u00ad\u2010Orbitrap\t\r \u00a0Several\t\r \u00a0types\t\r \u00a0of\t\r \u00a0mass\t\r \u00a0spectrometers\t\r \u00a0can\t\r \u00a0be\t\r \u00a0employed\t\r \u00a0for\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0experiments,\t\r \u00a0the\t\r \u00a0most\t\r \u00a0common\t\r \u00a0of\t\r \u00a0which\t\r \u00a0are\t\r \u00a0quadrupole\t\r \u00a0time-\u00ad\u2010of-\u00ad\u2010flight\t\r \u00a0instruments,\t\r \u00a0ion\t\r \u00a0traps,\t\r \u00a0and\t\r \u00a0the\t\r \u00a0newer\t\r \u00a0Orbitrap\t\r \u00a0and\t\r \u00a0FT-\u00ad\u2010ICR\t\r \u00a0mass\t\r \u00a0spectrometers(299-\u00ad\u2010302).\t\r \u00a0An\t\r \u00a0LTQ-\u00ad\u2010Orbitrap\t\r \u00a0was\t\r \u00a0used\t\r \u00a0exclusively\t\r \u00a0for\t\r \u00a0experiments\t\r \u00a0presented\t\r \u00a0here,\t\r \u00a0and\t\r \u00a0thus\t\r \u00a0protein\t\r \u00a0identification\t\r \u00a0is\t\r \u00a0discussed\t\r \u00a0in\t\r \u00a0relation\t\r \u00a0to\t\r \u00a0this\t\r \u00a0instrument.\t\r \u00a0 \t\r \u00a0\t\r \u00a0 35\t\r \u00a0 An\t\r \u00a0LTQ-\u00ad\u2010Orbitrap\t\r \u00a0is\t\r \u00a0a\t\r \u00a0hybrid\t\r \u00a0instrument\t\r \u00a0consisting\t\r \u00a0of\t\r \u00a0a\t\r \u00a0linear\t\r \u00a0ion\t\r \u00a0trap\t\r \u00a0mass\t\r \u00a0spectrometer\t\r \u00a0coupled\t\r \u00a0to\t\r \u00a0an\t\r \u00a0orbitrap\t\r \u00a0mass\t\r \u00a0analyzer(303).\t\r \u00a0In\t\r \u00a0a\t\r \u00a0typical\t\r \u00a0proteomics\t\r \u00a0work\t\r \u00a0flow,\t\r \u00a0peptides\t\r \u00a0are\t\r \u00a0resolved\t\r \u00a0on\t\r \u00a0a\t\r \u00a0nanoscale\t\r \u00a0reversed\t\r \u00a0phase\t\r \u00a0HPLC\t\r \u00a0column\t\r \u00a0with\t\r \u00a0an\t\r \u00a0inner\t\r \u00a0diameter\t\r \u00a0and\t\r \u00a0flow\t\r \u00a0rates\t\r \u00a0of\t\r \u00a0approximately\t\r \u00a075\u00b5m\t\r \u00a0and\t\r \u00a0200nL\/min\t\r \u00a0respectively,\t\r \u00a0and\t\r \u00a0a\t\r \u00a0C18\t\r \u00a0stationary\t\r \u00a0phase\t\r \u00a0and\t\r \u00a0mobile\t\r \u00a0phase\t\r \u00a0consisting\t\r \u00a0of\t\r \u00a0increasing\t\r \u00a0concentrations\t\r \u00a0of\t\r \u00a0acetonitrile\t\r \u00a0at\t\r \u00a0low\t\r \u00a0pH(304).\t\r \u00a0Peptides\t\r \u00a0eluting\t\r \u00a0from\t\r \u00a0the\t\r \u00a0LC\t\r \u00a0are\t\r \u00a0ionized\t\r \u00a0by\t\r \u00a0an\t\r \u00a0electrospray\t\r \u00a0source\t\r \u00a0and\t\r \u00a0focused\t\r \u00a0through\t\r \u00a0a\t\r \u00a0series\t\r \u00a0of\t\r \u00a0two\t\r \u00a0quadrupoles\t\r \u00a0into\t\r \u00a0the\t\r \u00a0linear\t\r \u00a0ion\t\r \u00a0trap.\t\r \u00a0Automatic\t\r \u00a0gain\t\r \u00a0control\t\r \u00a0(AGC)\t\r \u00a0is\t\r \u00a0then\t\r \u00a0employed,\t\r \u00a0which\t\r \u00a0entails\t\r \u00a0a\t\r \u00a0short\t\r \u00a0pre-\u00ad\u2010scan\t\r \u00a0within\t\r \u00a0the\t\r \u00a0ion\t\r \u00a0trap\t\r \u00a0used\t\r \u00a0to\t\r \u00a0enable\t\r \u00a0storage\t\r \u00a0of\t\r \u00a0a\t\r \u00a0defined\t\r \u00a0number\t\r \u00a0of\t\r \u00a0ions(300).\t\r \u00a0Ions\t\r \u00a0within\t\r \u00a0a\t\r \u00a0m\/z\t\r \u00a0range\t\r \u00a0of\t\r \u00a0~300-\u00ad\u20101600\t\r \u00a0are\t\r \u00a0then\t\r \u00a0passed\t\r \u00a0into\t\r \u00a0the\t\r \u00a0c-\u00ad\u2010trap,\t\r \u00a0which\t\r \u00a0is\t\r \u00a0a\t\r \u00a0curved\t\r \u00a0quadrupole\t\r \u00a0responsible\t\r \u00a0for\t\r \u00a0collecting\t\r \u00a0and\t\r \u00a0storing\t\r \u00a0ions\t\r \u00a0prior\t\r \u00a0to\t\r \u00a0pulsed\t\r \u00a0injection\t\r \u00a0into\t\r \u00a0the\t\r \u00a0orbitrap.\t\r \u00a0Ions\t\r \u00a0are\t\r \u00a0injected\t\r \u00a0into\t\r \u00a0the\t\r \u00a0orbitrap\t\r \u00a0perpendicular\t\r \u00a0to\t\r \u00a0an\t\r \u00a0electromagnetic\t\r \u00a0field,\t\r \u00a0and\t\r \u00a0at\t\r \u00a0a\t\r \u00a0point\t\r \u00a0offset\t\r \u00a0from\t\r \u00a0centre\t\r \u00a0along\t\r \u00a0spindle-\u00ad\u2010shaped\t\r \u00a0electrode,\t\r \u00a0which\t\r \u00a0results\t\r \u00a0in\t\r \u00a0harmonic\t\r \u00a0oscillations(301).\t\r \u00a0Packets\t\r \u00a0of\t\r \u00a0ions\t\r \u00a0with\t\r \u00a0a\t\r \u00a0particular\t\r \u00a0m\/z\t\r \u00a0ratio\t\r \u00a0oscillate\t\r \u00a0together\t\r \u00a0as\t\r \u00a0rings,\t\r \u00a0and\t\r \u00a0the\t\r \u00a0frequency\t\r \u00a0of\t\r \u00a0these\t\r \u00a0oscillations\t\r \u00a0is\t\r \u00a0related\t\r \u00a0to\t\r \u00a0their\t\r \u00a0m\/z\t\r \u00a0ratio.\t\r \u00a0While\t\r \u00a0slower\t\r \u00a0and\t\r \u00a0not\t\r \u00a0as\t\r \u00a0sensitive\t\r \u00a0as\t\r \u00a0a\t\r \u00a0linear\t\r \u00a0ion\t\r \u00a0trap,\t\r \u00a0the\t\r \u00a0orbitrap\t\r \u00a0is\t\r \u00a0capable\t\r \u00a0of\t\r \u00a0resolutions\t\r \u00a0as\t\r \u00a0high\t\r \u00a0as\t\r \u00a0150,000\t\r \u00a0and,\t\r \u00a0when\t\r \u00a0lock-\u00ad\u2010mass\t\r \u00a0is\t\r \u00a0employed\t\r \u00a0for\t\r \u00a0real\t\r \u00a0time\t\r \u00a0calibration,\t\r \u00a0sub\t\r \u00a0parts\t\r \u00a0per\t\r \u00a0million\t\r \u00a0mass\t\r \u00a0accuracy(303,\t\r \u00a0305).\t\r \u00a0In\t\r \u00a0a\t\r \u00a0typical\t\r \u00a0LTQ-\u00ad\u2010Orbitrap\t\r \u00a0configuration,\t\r \u00a0a\t\r \u00a0scan\t\r \u00a0termed\t\r \u00a0an\t\r \u00a0MS\t\r \u00a0or\t\r \u00a0survey\t\r \u00a0scan\t\r \u00a0(as\t\r \u00a0described\t\r \u00a0above)\t\r \u00a0is\t\r \u00a0initially\t\r \u00a0collected\t\r \u00a0within\t\r \u00a0the\t\r \u00a0Orbitrap,\t\r \u00a0and\t\r \u00a0determines\t\r \u00a0the\t\r \u00a0m\/z\t\r \u00a0of\t\r \u00a0all\t\r \u00a0peptides\t\r \u00a0currently\t\r 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\u00a0way\t\r \u00a0at\t\r \u00a0the\t\r \u00a0protein\t\r \u00a0level(315,\t\r \u00a0316).\t\r \u00a0\t\r \u00a0 1.2.5\t\r \u00a0Quantitation\t\r \u00a0Due\t\r \u00a0to\t\r \u00a0the\t\r \u00a0dynamic\t\r \u00a0nature\t\r \u00a0of\t\r \u00a0proteins,\t\r \u00a0the\t\r \u00a0ability\t\r \u00a0to\t\r \u00a0quantify\t\r \u00a0protein\t\r \u00a0levels\t\r \u00a0between\t\r \u00a0various\t\r \u00a0samples\t\r \u00a0is\t\r \u00a0an\t\r \u00a0important\t\r \u00a0aspect\t\r \u00a0of\t\r \u00a0proteomics(318,\t\r \u00a0319).\t\r \u00a0This\t\r \u00a0can\t\r \u00a0be\t\r \u00a0done\t\r \u00a0in\t\r \u00a0two\t\r \u00a0ways.\t\r \u00a0Most\t\r \u00a0often,\t\r \u00a0stable\t\r \u00a0isotope\t\r \u00a0labels\t\r \u00a0are\t\r \u00a0incorporated\t\r \u00a0into\t\r \u00a0proteins\t\r \u00a0or\t\r \u00a0peptides.\t\r \u00a0Because\t\r \u00a0these\t\r \u00a0isotopologs\t\r \u00a0are\t\r \u00a0identical\t\r \u00a0in\t\r \u00a0their\t\r \u00a0chemical\t\r \u00a0properties,\t\r \u00a0they\t\r 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\u00a0number\t\r \u00a0of\t\r \u00a0MSn\t\r \u00a0spectra\t\r \u00a0generated\t\r \u00a0by\t\r \u00a0peptides\t\r \u00a0across\t\r \u00a0various\t\r \u00a0samples.\t\r \u00a0While\t\r \u00a0several\t\r \u00a0advantages\t\r \u00a0such\t\r \u00a0as\t\r \u00a0reduced\t\r \u00a0cost\t\r \u00a0and\t\r \u00a0sample\t\r \u00a0handling,\t\r \u00a0avoiding\t\r \u00a0increased\t\r \u00a0spectral\t\r \u00a0complexity\t\r \u00a0caused\t\r \u00a0by\t\r \u00a0isotopic\t\r \u00a0labels,\t\r \u00a0and\t\r \u00a0potentially\t\r \u00a0higher\t\r \u00a0dynamic\t\r \u00a0range\t\r \u00a0capabilities\t\r \u00a0have\t\r \u00a0been\t\r \u00a0reported,\t\r \u00a0label\t\r \u00a0free\t\r \u00a0methods\t\r \u00a0are\t\r \u00a0less\t\r \u00a0accurate,\t\r \u00a0are\t\r \u00a0becoming\t\r \u00a0much\t\r \u00a0less\t\r \u00a0widely\t\r \u00a0used,\t\r \u00a0and\t\r \u00a0will\t\r \u00a0not\t\r \u00a0be\t\r \u00a0discussed\t\r \u00a0further\t\r \u00a0here(320-\u00ad\u2010323).\t\r \u00a0The\t\r \u00a0earliest\t\r \u00a0point\t\r \u00a0at\t\r \u00a0which\t\r \u00a0an\t\r \u00a0isotopic\t\r \u00a0label\t\r \u00a0can\t\r \u00a0be\t\r \u00a0introduced\t\r \u00a0into\t\r \u00a0a\t\r \u00a0sample\t\r \u00a0is\t\r \u00a0by\t\r \u00a0metabolic\t\r \u00a0labeling\t\r \u00a0during\t\r \u00a0cell\t\r \u00a0growth\t\r \u00a0or\t\r \u00a0division.\t\r \u00a0The\t\r \u00a0first\t\r \u00a0reports\t\r \u00a0of\t\r \u00a0metabolic\t\r \u00a0labeling\t\r \u00a0were\t\r \u00a0described\t\r \u00a0in\t\r \u00a0bacteria\t\r \u00a0using\t\r \u00a015N-\u00ad\u2010enriched\t\r \u00a0media(324).\t\r \u00a0Subsequently,\t\r \u00a0 \t\r \u00a0\t\r \u00a0 39\t\r \u00a0 this\t\r \u00a0method\t\r \u00a0has\t\r \u00a0been\t\r \u00a0described\t\r \u00a0for\t\r \u00a0other\t\r \u00a0organisms\t\r \u00a0such\t\r \u00a0as\t\r \u00a0C.\t\r \u00a0elegans,\t\r \u00a0Drosophila,\t\r \u00a0and\t\r \u00a0Arabidopsis(325-\u00ad\u2010328).\t\r \u00a0However,\t\r \u00a0challenges\t\r \u00a0associated\t\r \u00a0with\t\r \u00a0this\t\r \u00a0system\t\r \u00a0arise\t\r \u00a0due\t\r \u00a0to\t\r \u00a0variable\t\r \u00a0mass\t\r \u00a0shifts\t\r \u00a0between\t\r \u00a0unlabeled\t\r \u00a0and\t\r \u00a0labeled\t\r \u00a0peptides\t\r \u00a0and\t\r \u00a0thus,\t\r \u00a0data\t\r \u00a0analysis\t\r \u00a0can\t\r \u00a0be\t\r \u00a0challenging.\t\r \u00a0An\t\r \u00a0alternative\t\r \u00a0to\t\r \u00a015N-\u00ad\u2010labeling\t\r \u00a0is\t\r \u00a0Stable\t\r \u00a0isotope\t\r \u00a0labeling\t\r \u00a0by\t\r \u00a0amino\t\r \u00a0acids\t\r \u00a0in\t\r \u00a0cell\t\r \u00a0culture\t\r \u00a0(SILAC),\t\r \u00a0which\t\r \u00a0involves\t\r \u00a0growing\t\r \u00a0tissue\t\r \u00a0culture\t\r \u00a0cells\t\r \u00a0in\t\r \u00a0media\t\r \u00a0supplemented\t\r \u00a0with\t\r \u00a0isotopically\t\r \u00a0labeled\t\r \u00a0forms\t\r \u00a0of\t\r \u00a0lysine\t\r \u00a0and\t\r \u00a0arginine(297,\t\r \u00a0329-\u00ad\u2010331).\t\r \u00a0Typically\t\r \u00a0greater\t\r \u00a0than\t\r \u00a090%\t\r \u00a0protein\t\r \u00a0labeling\t\r \u00a0is\t\r \u00a0achieved\t\r \u00a0after\t\r \u00a06-\u00ad\u20108\t\r \u00a0cell\t\r \u00a0passages,\t\r \u00a0and\t\r \u00a0tryptic\t\r \u00a0digestion\t\r \u00a0results\t\r \u00a0in\t\r \u00a0at\t\r \u00a0least\t\r \u00a0one\t\r \u00a0label\t\r \u00a0per\t\r \u00a0peptide\t\r \u00a0(>1\t\r \u00a0in\t\r \u00a0cases\t\r \u00a0of\t\r \u00a0missed\t\r \u00a0cleavages),\t\r \u00a0except\t\r \u00a0for\t\r \u00a0the\t\r \u00a0very\t\r \u00a0c-\u00ad\u2010terminal\t\r \u00a0peptide,\t\r \u00a0which\t\r \u00a0cannot\t\r \u00a0be\t\r \u00a0labeled.\t\r \u00a0Thus,\t\r \u00a0an\t\r \u00a0advantage\t\r \u00a0of\t\r \u00a0SILAC\t\r \u00a0is\t\r \u00a0that\t\r \u00a0the\t\r \u00a0number\t\r \u00a0of\t\r \u00a0incorporated\t\r \u00a0labels\t\r \u00a0is\t\r \u00a0defined\t\r \u00a0and\t\r \u00a0does\t\r \u00a0not\t\r \u00a0depend\t\r \u00a0on\t\r \u00a0peptide\t\r \u00a0sequence,\t\r \u00a0which\t\r \u00a0greatly\t\r \u00a0simplifies\t\r \u00a0analysis.\t\r \u00a0Some\t\r \u00a0limitations\t\r \u00a0to\t\r \u00a0SILAC\t\r \u00a0include\t\r \u00a0a\t\r \u00a0maximum\t\r \u00a0of\t\r \u00a0three\t\r \u00a0isotopically\t\r \u00a0labeled\t\r \u00a0populations\t\r \u00a0per\t\r \u00a0experiment,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0the\t\r \u00a0fact\t\r \u00a0that\t\r \u00a0some\t\r \u00a0cell\t\r \u00a0lines\t\r \u00a0aren\u2019t\t\r \u00a0amenable\t\r \u00a0to\t\r \u00a0metabolic\t\r \u00a0labeling,\t\r \u00a0and\t\r \u00a0it\t\r \u00a0is\t\r \u00a0not\t\r \u00a0known\t\r \u00a0how\t\r \u00a0or\t\r \u00a0if\t\r \u00a0cells\t\r \u00a0are\t\r \u00a0affected\t\r \u00a0by\t\r \u00a0the\t\r \u00a0labeled\t\r \u00a0amino\t\r \u00a0acids.\t\r \u00a0However,\t\r \u00a0SILAC\t\r \u00a0has\t\r \u00a0been\t\r \u00a0applied\t\r \u00a0extensively\t\r \u00a0in\t\r \u00a0proteomics\t\r \u00a0applications\t\r \u00a0ranging\t\r \u00a0from\t\r \u00a0expression\t\r \u00a0profiling,\t\r \u00a0cell\t\r \u00a0signaling\t\r \u00a0and\t\r \u00a0identification\t\r \u00a0of\t\r \u00a0protein\t\r \u00a0complexes(332-\u00ad\u2010337).\t\r \u00a0Recently\t\r \u00a0the\t\r \u00a0SILAC\t\r \u00a0labeled\t\r \u00a0mouse\t\r \u00a0was\t\r \u00a0also\t\r \u00a0introduced,\t\r \u00a0opening\t\r \u00a0the\t\r \u00a0door\t\r \u00a0to\t\r \u00a0a\t\r \u00a0plethora\t\r \u00a0of\t\r \u00a0new\t\r \u00a0in\t\r \u00a0vivo\t\r \u00a0applications(338).\t\r \u00a0Several\t\r \u00a0chemical\t\r \u00a0labeling\t\r \u00a0methods\t\r \u00a0have\t\r \u00a0been\t\r \u00a0derived\t\r \u00a0which\t\r \u00a0target\t\r \u00a0specific\t\r \u00a0reactive\t\r \u00a0sites\t\r \u00a0on\t\r \u00a0proteins\t\r \u00a0or\t\r \u00a0peptides\t\r \u00a0for\t\r \u00a0incorporation\t\r \u00a0of\t\r \u00a0isotopic\t\r \u00a0labels(318,\t\r \u00a0319).\t\r \u00a0The\t\r \u00a0three\t\r \u00a0most\t\r \u00a0widely\t\r \u00a0applied\t\r \u00a0are\t\r \u00a0discussed\t\r \u00a0here.\t\r \u00a0Initially,\t\r \u00a0Isotope-\u00ad\u2010coded\t\r \u00a0affinity\t\r \u00a0tagging\t\r \u00a0(iCAT)\t\r \u00a0was\t\r \u00a0developed\t\r \u00a0which\t\r \u00a0targets\t\r \u00a0cysteine\t\r \u00a0residues\t\r \u00a0with\t\r \u00a0a\t\r \u00a0tag\t\r \u00a0consisting\t\r \u00a0of\t\r \u00a0a\t\r \u00a0reactive\t\r \u00a0iodoacetamide\t\r \u00a0group,\t\r \u00a0a\t\r \u00a0linker\t\r \u00a0group\t\r \u00a0either\t\r \u00a0light\t\r \u00a0labeled\t\r \u00a0or\t\r \u00a0containing\t\r \u00a0 \t\r \u00a0\t\r \u00a0 40\t\r \u00a0 nine\t\r \u00a013C\t\r \u00a0atoms,\t\r \u00a0and\t\r \u00a0a\t\r \u00a0cleavable\t\r \u00a0biotin\t\r \u00a0tag\t\r \u00a0for\t\r \u00a0purification(339-\u00ad\u2010341).\t\r \u00a0In\t\r \u00a0a\t\r \u00a0typical\t\r \u00a0experiment\t\r \u00a0reduced\t\r \u00a0and\t\r \u00a0denatured\t\r \u00a0proteins\t\r \u00a0are\t\r \u00a0modified\t\r \u00a0with\t\r \u00a0either\t\r \u00a0a\t\r \u00a0light\t\r \u00a0or\t\r \u00a0heavy\t\r \u00a0iCAT\t\r \u00a0tag,\t\r \u00a0trypsinized,\t\r \u00a0and\t\r \u00a0tagged\t\r \u00a0peptides\t\r \u00a0are\t\r \u00a0isolated\t\r \u00a0on\t\r \u00a0an\t\r \u00a0avidin\t\r \u00a0column.\t\r \u00a0Due\t\r \u00a0to\t\r \u00a0specific\t\r \u00a0targeting\t\r \u00a0of\t\r \u00a0cysteine\t\r \u00a0residues,\t\r \u00a0iCAT\t\r \u00a0has\t\r \u00a0been\t\r \u00a0reported\t\r \u00a0to\t\r \u00a0reduce\t\r \u00a0sample\t\r \u00a0complexity.\t\r \u00a0However\t\r \u00a0this\t\r \u00a0also\t\r \u00a0means\t\r \u00a0that\t\r \u00a0proteins\t\r \u00a0void\t\r \u00a0or\t\r \u00a0sparse\t\r \u00a0in\t\r \u00a0cysteine\t\r \u00a0residues\t\r \u00a0are\t\r \u00a0identified\t\r \u00a0by\t\r \u00a0only\t\r \u00a0a\t\r \u00a0few\t\r \u00a0peptides\t\r \u00a0or\t\r \u00a0not\t\r \u00a0at\t\r \u00a0all,\t\r \u00a0and\t\r \u00a0it\t\r \u00a0is\t\r \u00a0not\t\r \u00a0well\t\r \u00a0suited\t\r \u00a0for\t\r \u00a0PTM\t\r \u00a0analysis,\t\r \u00a0except\t\r \u00a0for\t\r \u00a0specialized\t\r \u00a0applications\t\r \u00a0such\t\r \u00a0as\t\r \u00a0for\t\r \u00a0studying\t\r \u00a0cysteine\t\r \u00a0proteases.\t\r \u00a0Two\t\r \u00a0additional\t\r \u00a0chemical\t\r \u00a0labeling\t\r \u00a0techniques,\t\r \u00a0iTRAQ\t\r \u00a0and\t\r \u00a0dimethylation\t\r \u00a0or\t\r \u00a0formaldehyde\t\r \u00a0labeling,\t\r \u00a0both\t\r \u00a0target\t\r \u00a0\u03b1-\u00ad\u2010\t\r \u00a0and\t\r \u00a0\u03b5-\u00ad\u2010amino\t\r \u00a0groups.\t\r \u00a0Formaldehyde\t\r \u00a0labeling\t\r \u00a0involves\t\r \u00a0dimethylation\t\r \u00a0of\t\r \u00a0these\t\r \u00a0groups\t\r \u00a0through\t\r \u00a0reductive\t\r \u00a0amination,\t\r \u00a0and\t\r \u00a0is\t\r \u00a0an\t\r \u00a0appealing\t\r \u00a0labeling\t\r \u00a0technique\t\r \u00a0in\t\r \u00a0that\t\r \u00a0it\t\r \u00a0is\t\r \u00a0very\t\r \u00a0fast,\t\r \u00a0specific\t\r \u00a0and\t\r \u00a0cheap(342).\t\r \u00a0Furthermore,\t\r \u00a0three\t\r \u00a0forms\t\r \u00a0exist,\t\r \u00a0enabling\t\r \u00a0tri-\u00ad\u2010plex\t\r \u00a0quantitation(343).\t\r \u00a0One\t\r \u00a0drawback\t\r \u00a0of\t\r \u00a0dimethylation\t\r \u00a0is\t\r \u00a0that\t\r \u00a0it\t\r \u00a0relies\t\r \u00a0of\t\r \u00a0deuterated\t\r \u00a0labels,\t\r \u00a0which\t\r \u00a0can\t\r \u00a0introduce\t\r \u00a0elution\t\r \u00a0shifts\t\r \u00a0during\t\r \u00a0LC.\t\r \u00a0iTRAQ\t\r \u00a0(Isotope\t\r \u00a0tags\t\r \u00a0for\t\r \u00a0relative\t\r \u00a0and\t\r \u00a0absolute\t\r \u00a0quantification)\t\r \u00a0is\t\r \u00a0similar\t\r \u00a0to\t\r \u00a0iCAT\t\r \u00a0in\t\r \u00a0that\t\r \u00a0peptides\t\r \u00a0are\t\r \u00a0labeled\t\r \u00a0with\t\r \u00a0isobaric\t\r \u00a0tags,\t\r \u00a0which\t\r \u00a0facilitate\t\r \u00a0the\t\r \u00a0relative\t\r \u00a0comparison\t\r \u00a0of\t\r \u00a0samples(344).\t\r \u00a0However,\t\r \u00a0unique\t\r \u00a0to\t\r \u00a0iTRAQ,\t\r \u00a0these\t\r \u00a0tags\t\r \u00a0are\t\r \u00a0indistinguishable\t\r \u00a0in\t\r \u00a0the\t\r \u00a0MS\t\r \u00a0mode\t\r \u00a0and\t\r \u00a0quantitation\t\r \u00a0is\t\r \u00a0achieved\t\r \u00a0in\t\r \u00a0the\t\r \u00a0MSn\t\r \u00a0mode\t\r 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\u00a0allow\t\r \u00a0accurate\t\r \u00a0quantitation.\t\r \u00a0Finally,\t\r \u00a0several\t\r \u00a0software\t\r \u00a0applications\t\r \u00a0have\t\r \u00a0been\t\r \u00a0developed\t\r \u00a0for\t\r \u00a0quantitative\t\r \u00a0mass\t\r \u00a0spectrometry-\u00ad\u2010based\t\r \u00a0proteomics(350,\t\r \u00a0351).\t\r \u00a0\t\r \u00a0 1.2.6\t\r \u00a0Pre-\u00ad\u2010fractionation\t\r \u00a0and\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0PTMs\t\r \u00a0One\t\r \u00a0undeniable\t\r \u00a0shortcoming\t\r \u00a0of\t\r \u00a0current\t\r \u00a0proteomics\t\r \u00a0approaches\t\r \u00a0is\t\r \u00a0that\t\r \u00a0we\t\r \u00a0are\t\r \u00a0not\t\r \u00a0yet\t\r \u00a0able\t\r \u00a0to\t\r \u00a0achieve\t\r \u00a0full\t\r \u00a0proteome\t\r \u00a0coverage.\t\r \u00a0This\t\r \u00a0is\t\r \u00a0a\t\r \u00a0daunting\t\r \u00a0task\t\r \u00a0however,\t\r \u00a0as\t\r \u00a0of\t\r \u00a0the\t\r \u00a0estimated\t\r \u00a020,500\t\r \u00a0protein-\u00ad\u2010coding\t\r \u00a0genes\t\r \u00a0in\t\r \u00a0humans,\t\r \u00a010,000\t\r \u00a0are\t\r \u00a0estimated\t\r \u00a0to\t\r \u00a0be\t\r \u00a0expressed\t\r \u00a0per\t\r \u00a0cell\t\r \u00a0at\t\r \u00a0any\t\r \u00a0given\t\r \u00a0time,\t\r \u00a0a\t\r \u00a0number\t\r \u00a0that\t\r \u00a0becomes\t\r \u00a0much\t\r \u00a0greater\t\r \u00a0when\t\r \u00a0PTMs\t\r \u00a0are\t\r \u00a0considered(352,\t\r \u00a0353).\t\r \u00a0With\t\r \u00a0a\t\r \u00a0few\t\r \u00a0exceptions,\t\r \u00a0large-\u00ad\u2010scale\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0analyses\t\r \u00a0detect\t\r \u00a020%\t\r \u00a0of\t\r \u00a0these\t\r \u00a0at\t\r \u00a0best.\t\r \u00a0In\t\r \u00a0addition,\t\r \u00a0the\t\r \u00a0dynamic\t\r \u00a0range\t\r \u00a0in\t\r \u00a0biological\t\r \u00a0samples\t\r \u00a0can\t\r \u00a0be\t\r \u00a0as\t\r \u00a0high\t\r \u00a0as\t\r \u00a012\t\r \u00a0orders\t\r \u00a0of\t\r \u00a0magnitude,\t\r \u00a0while\t\r \u00a0current\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0systems\t\r \u00a0are\t\r \u00a0capable\t\r \u00a0of\t\r \u00a0only\t\r \u00a04,\t\r \u00a0meaning\t\r \u00a0that\t\r \u00a0considerable\t\r \u00a0progress\t\r \u00a0is\t\r \u00a0required\t\r \u00a0to\t\r \u00a0detect\t\r \u00a0low\t\r \u00a0abundance\t\r \u00a0proteins(301,\t\r \u00a0302,\t\r \u00a0354,\t\r \u00a0355).\t\r \u00a0To\t\r \u00a0improve\t\r \u00a0sample\t\r \u00a0coverage,\t\r \u00a0most\t\r \u00a0proteomics\t\r \u00a0workflows\t\r \u00a0include\t\r \u00a0at\t\r \u00a0least\t\r \u00a0one\t\r \u00a0of\t\r \u00a0a\t\r \u00a0number\t\r \u00a0of\t\r \u00a0subfractionation\t\r \u00a0steps\t\r \u00a0upstream\t\r \u00a0of\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0to\t\r \u00a0reduce\t\r \u00a0sample\t\r \u00a0complexity.\t\r \u00a0Such\t\r \u00a0techniques\t\r \u00a0range\t\r \u00a0from\t\r \u00a0orthogonal\t\r \u00a0LC\t\r \u00a0techniques\t\r \u00a0such\t\r \u00a0as\t\r \u00a0strong\t\r \u00a0cation\t\r \u00a0and\t\r \u00a0strong\t\r \u00a0anion\t\r \u00a0exchange\t\r \u00a0(SCX\t\r \u00a0and\t\r \u00a0SAX),\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0size\t\r \u00a0exclusion\t\r \u00a0(SEX)\t\r \u00a0chromatography,\t\r \u00a0isoelectric\t\r \u00a0focusing\t\r \u00a0(IEF),\t\r \u00a0and\t\r \u00a0sodium\t\r \u00a0dodecyl\t\r \u00a0sulfate-\u00ad\u2010polyacrylamide\t\r \u00a0gel\t\r \u00a0electrophoresis\t\r \u00a0(SDS-\u00ad\u2010PAGE)(356-\u00ad\u2010363).\t\r \u00a0While\t\r \u00a0little\t\r \u00a0work\t\r \u00a0has\t\r \u00a0 \t\r \u00a0\t\r \u00a0 42\t\r \u00a0 been\t\r \u00a0done\t\r \u00a0to\t\r \u00a0compare\t\r \u00a0the\t\r \u00a0effectiveness\t\r \u00a0of\t\r \u00a0these\t\r \u00a0techniques,\t\r \u00a0it\t\r \u00a0was\t\r \u00a0recently\t\r \u00a0shown\t\r \u00a0that\t\r \u00a0among\t\r \u00a0the\t\r \u00a0most\t\r \u00a0commonly\t\r \u00a0used\t\r \u00a0(SCX,\t\r \u00a0IEF\t\r \u00a0and\t\r \u00a0SDS-\u00ad\u2010PAGE),\t\r \u00a0SDS-\u00ad\u2010PAGE\t\r \u00a0is\t\r \u00a0the\t\r \u00a0preferred\t\r \u00a0technique\t\r \u00a0in\t\r \u00a0terms\t\r \u00a0of\t\r \u00a0proteome\t\r \u00a0coverage,\t\r \u00a0while\t\r \u00a0IEF\t\r \u00a0and\t\r \u00a0SCX\t\r \u00a0offer\t\r \u00a0superior\t\r \u00a0sample\t\r \u00a0recovery(364).\t\r \u00a0IEF\t\r \u00a0has\t\r \u00a0also\t\r \u00a0been\t\r \u00a0reported\t\r \u00a0to\t\r \u00a0offer\t\r \u00a0superior\t\r \u00a0reproducibility\t\r \u00a0and\t\r \u00a0resolution\t\r \u00a0as\t\r \u00a0compared\t\r \u00a0to\t\r \u00a0SCX(365).\t\r \u00a0Subcellular\t\r \u00a0fractionation\t\r \u00a0steps\t\r \u00a0are\t\r \u00a0also\t\r \u00a0very\t\r \u00a0advantageous\t\r \u00a0for\t\r \u00a0sub-\u00ad\u2010proteome\t\r \u00a0studies,\t\r \u00a0such\t\r \u00a0as\t\r \u00a0those\t\r \u00a0addressing\t\r \u00a0protein\t\r \u00a0complexes,\t\r \u00a0particular\t\r \u00a0subcellular\t\r \u00a0compartments,\t\r \u00a0and\t\r \u00a0post-\u00ad\u2010translational\t\r \u00a0modifications.\t\r \u00a0While\t\r \u00a0traditional\t\r \u00a0biochemical\t\r \u00a0purification\t\r \u00a0techniques\t\r \u00a0such\t\r \u00a0as\t\r \u00a0immunoprecipitation\t\r \u00a0and\t\r \u00a0density\t\r \u00a0gradients\t\r \u00a0have\t\r \u00a0been\t\r \u00a0applied\t\r \u00a0to\t\r \u00a0the\t\r \u00a0former(296,\t\r \u00a0337,\t\r \u00a0366-\u00ad\u2010369),\t\r \u00a0several\t\r \u00a0additional\t\r \u00a0techniques\t\r \u00a0have\t\r \u00a0been\t\r \u00a0developed\t\r \u00a0to\t\r \u00a0analyze\t\r \u00a0PTMs.\t\r \u00a0\t\r \u00a0Due\t\r \u00a0to\t\r \u00a0the\t\r \u00a0fact\t\r \u00a0that\t\r \u00a0they\t\r \u00a0are\t\r \u00a0generally\t\r \u00a0substoichiometric\t\r \u00a0and\t\r \u00a0targeted\t\r \u00a0to\t\r \u00a0only\t\r \u00a0specific\t\r \u00a0residues\t\r \u00a0within\t\r \u00a0a\t\r \u00a0protein\t\r \u00a0sequence,\t\r \u00a0peptides\t\r \u00a0containing\t\r \u00a0a\t\r \u00a0specific\t\r \u00a0PTM\t\r \u00a0generally\t\r \u00a0need\t\r \u00a0to\t\r \u00a0be\t\r \u00a0enriched\t\r \u00a0prior\t\r \u00a0to\t\r \u00a0LC-\u00ad\u2010MSn.\t\r \u00a0Several\t\r \u00a0enrichment\t\r \u00a0techniques\t\r \u00a0exist,\t\r \u00a0including\t\r \u00a0immuno-\u00ad\u2010enrichment,\t\r \u00a0in\t\r \u00a0vitro\t\r \u00a0chemical\t\r \u00a0tagging,\t\r \u00a0enzymatic\t\r \u00a0labeling,\t\r \u00a0ion\t\r \u00a0interactions,\t\r \u00a0and\t\r \u00a0other\t\r \u00a0approaches\t\r \u00a0such\t\r \u00a0as\t\r \u00a0lectin\t\r \u00a0affinity\t\r \u00a0chromatography.\t\r \u00a0Examples\t\r \u00a0of\t\r \u00a0immuno-\u00ad\u2010enrichment\t\r \u00a0include\t\r \u00a0phosphorylated\t\r \u00a0tyrosine,\t\r \u00a0lysine\t\r \u00a0acetylation\t\r \u00a0and\t\r \u00a0arginine\t\r \u00a0methylation\t\r \u00a0specific\t\r \u00a0antibodies,\t\r \u00a0while\t\r \u00a0\u03b2-\u00ad\u2010elimination\t\r \u00a0and\t\r \u00a0subsequent\t\r \u00a0incorporation\t\r \u00a0of\t\r \u00a0an\t\r \u00a0affinity-\u00ad\u2010tag\t\r \u00a0has\t\r \u00a0been\t\r \u00a0described\t\r \u00a0for\t\r \u00a0phosphorylated\t\r \u00a0and\t\r \u00a0O-\u00ad\u2010GlcNAcylated\t\r \u00a0serine\t\r \u00a0and\t\r \u00a0threonine\t\r \u00a0residues(331,\t\r \u00a0370-\u00ad\u2010373).\t\r \u00a0Phospholipases\t\r \u00a0have\t\r \u00a0been\t\r \u00a0used\t\r \u00a0to\t\r \u00a0cleave\t\r \u00a0and\t\r \u00a0purify\t\r \u00a0glycosylphosphatidylinositol-\u00ad\u2010anchored\t\r \u00a0proteins\t\r \u00a0(GPI-\u00ad\u2010APs),\t\r \u00a0and\t\r \u00a0ionic\t\r \u00a0interactions\t\r \u00a0have\t\r \u00a0been\t\r \u00a0used\t\r \u00a0to\t\r \u00a0enrich\t\r \u00a0phosphorylated\t\r \u00a0serine,\t\r \u00a0threonine\t\r \u00a0and\t\r \u00a0tyrosine\t\r \u00a0residues(374-\u00ad\u2010376).\t\r \u00a0In\t\r \u00a0addition,\t\r \u00a0affinity-\u00ad\u2010tagged\t\r \u00a0ubiquitin\t\r \u00a0has\t\r \u00a0 \t\r \u00a0\t\r \u00a0 43\t\r \u00a0 been\t\r \u00a0expressed\t\r \u00a0in\t\r \u00a0cells\t\r \u00a0and\t\r \u00a0used\t\r \u00a0to\t\r \u00a0isolate\t\r \u00a0ubiquitylated\t\r \u00a0proteins,\t\r \u00a0and\t\r \u00a0N-\u00ad\u2010terminal\t\r \u00a0tryptic\t\r \u00a0peptides\t\r \u00a0have\t\r \u00a0been\t\r \u00a0isolated\t\r \u00a0to\t\r \u00a0identify\t\r \u00a0protease\t\r \u00a0substrates\t\r \u00a0and\t\r \u00a0cleavage\t\r \u00a0sites(377,\t\r \u00a0378).\t\r \u00a0In\t\r \u00a0each\t\r \u00a0case\t\r \u00a0where\t\r \u00a0specific\t\r \u00a0enrichment\t\r \u00a0has\t\r \u00a0been\t\r \u00a0achieved,\t\r \u00a0the\t\r \u00a0purified\t\r \u00a0proteins\t\r \u00a0or\t\r \u00a0peptides\t\r \u00a0are\t\r \u00a0analyzed\t\r \u00a0by\t\r \u00a0LC-\u00ad\u2010MSn,\t\r \u00a0which\t\r \u00a0results\t\r \u00a0in\t\r \u00a0sequence\t\r \u00a0information\t\r \u00a0localizing\t\r \u00a0the\t\r \u00a0modified\t\r \u00a0site(379).\t\r \u00a0However,\t\r \u00a0numerous\t\r \u00a0challenges\t\r \u00a0also\t\r \u00a0exist\t\r \u00a0in\t\r \u00a0PTM\t\r \u00a0analysis,\t\r \u00a0including\t\r \u00a0low\t\r \u00a0antibody\t\r \u00a0or\t\r \u00a0affinity\t\r \u00a0matrix\t\r \u00a0specificities,\t\r \u00a0chemical\t\r \u00a0side\t\r \u00a0reactions,\t\r \u00a0and\t\r \u00a0labile\t\r \u00a0modifications\t\r \u00a0that\t\r \u00a0can\t\r \u00a0prevent\t\r \u00a0sufficient\t\r \u00a0fragmentation\t\r \u00a0and\t\r \u00a0result\t\r \u00a0in\t\r \u00a0low\t\r \u00a0quality\t\r \u00a0high\t\r \u00a0order\t\r \u00a0spectra.\t\r \u00a0\t\r \u00a0 1.3\t\r \u00a0Phosphoproteomics\t\r \u00a0 1.3.1\t\r \u00a0History\t\r \u00a0and\t\r \u00a0current\t\r \u00a0potential\t\r \u00a0In\t\r \u00a0eukaryotes,\t\r \u00a0protein\t\r \u00a0phosphorylation\t\r \u00a0involves\t\r \u00a0the\t\r \u00a0reversible\t\r \u00a0addition\t\r \u00a0of\t\r \u00a0a\t\r \u00a0phosphate\t\r \u00a0to\t\r \u00a0serine,\t\r \u00a0threonine\t\r \u00a0and\t\r \u00a0tyrosine\t\r \u00a0residues,\t\r \u00a0which\t\r \u00a0is\t\r \u00a0thought\t\r \u00a0to\t\r \u00a0be\t\r \u00a0a\t\r \u00a0widespread\t\r \u00a0PTM,\t\r \u00a0occurring\t\r \u00a0in\t\r \u00a0approximately\t\r \u00a0one\t\r \u00a0third\t\r \u00a0of\t\r \u00a0all\t\r \u00a0proteins\t\r \u00a0and\t\r \u00a0regulating\t\r \u00a0protein\t\r \u00a0structure,\t\r \u00a0localization,\t\r \u00a0activity,\t\r \u00a0and\t\r \u00a0stability(380).\t\r \u00a0Phosphorylation\t\r \u00a0on\t\r \u00a0these\t\r \u00a0residues\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0histidine\t\r \u00a0and\t\r \u00a0aspartic\t\r \u00a0acid\t\r \u00a0has\t\r \u00a0also\t\r \u00a0been\t\r \u00a0reported\t\r \u00a0in\t\r \u00a0prokaryotes\t\r \u00a0(i.e.\t\r \u00a0two-\u00ad\u2010component\t\r \u00a0regulatory\t\r \u00a0systems)(381-\u00ad\u2010384).\t\r \u00a0However,\t\r \u00a0they\t\r \u00a0are\t\r \u00a0thought\t\r \u00a0to\t\r \u00a0be\t\r \u00a0much\t\r \u00a0more\t\r \u00a0rare.\t\r \u00a0Protein\t\r \u00a0phosphorylation\t\r \u00a0events\t\r \u00a0were\t\r \u00a0traditionally\t\r \u00a0studied\t\r \u00a0with\t\r \u00a0a\t\r \u00a0great\t\r \u00a0deal\t\r \u00a0of\t\r \u00a0myopia,\t\r \u00a0studying\t\r \u00a0one\t\r \u00a0or\t\r \u00a0a\t\r \u00a0few\t\r \u00a0events\t\r \u00a0in\t\r \u00a0isolation\t\r \u00a0as\t\r \u00a0no\t\r \u00a0tools\t\r \u00a0were\t\r \u00a0available\t\r \u00a0for\t\r \u00a0more\t\r \u00a0global\t\r \u00a0approaches.\t\r \u00a0However,\t\r \u00a0techniques\t\r \u00a0in\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0have\t\r \u00a0now\t\r \u00a0enabled\t\r \u00a0simultaneous\t\r \u00a0identification\t\r \u00a0of\t\r \u00a0thousands\t\r \u00a0of\t\r \u00a0phosphorylated\t\r \u00a0proteins,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0the\t\r \u00a0exact\t\r \u00a0localization\t\r \u00a0of\t\r \u00a0phosphorylated\t\r \u00a0residues\t\r \u00a0using\t\r \u00a0higher\t\r \u00a0order\t\r \u00a0spectra.\t\r \u00a0When\t\r \u00a0 \t\r \u00a0\t\r \u00a0 44\t\r \u00a0 combined\t\r \u00a0with\t\r \u00a0quantitative\t\r \u00a0techniques,\t\r \u00a0resolution\t\r \u00a0of\t\r \u00a0phosphorylation\t\r \u00a0dynamics\t\r \u00a0and\t\r \u00a0comparisons\t\r \u00a0between\t\r \u00a0various\t\r \u00a0stimuli\t\r \u00a0is\t\r \u00a0also\t\r \u00a0attainable.\t\r \u00a0However,\t\r \u00a0protein\t\r \u00a0phosphorylation\t\r \u00a0often\t\r \u00a0occurs\t\r \u00a0at\t\r \u00a0very\t\r \u00a0low\t\r \u00a0stoichiometry\t\r \u00a0and\t\r \u00a0is\t\r \u00a0reversible\t\r \u00a0by\t\r \u00a0phosphatases,\t\r \u00a0thus\t\r \u00a0requiring\t\r \u00a0specific\t\r \u00a0lysis\t\r \u00a0and\t\r \u00a0pre-\u00ad\u2010fractionation\t\r \u00a0methods\t\r \u00a0and\t\r \u00a0highly\t\r \u00a0specific\t\r \u00a0enrichment\t\r \u00a0techniques\t\r \u00a0prior\t\r \u00a0to\t\r \u00a0LC-\u00ad\u2010MSn.\t\r \u00a0In\t\r \u00a0addition,\t\r \u00a0the\t\r \u00a0lability\t\r \u00a0of\t\r \u00a0the\t\r \u00a0phosphate\t\r \u00a0during\t\r \u00a0CID\t\r \u00a0has\t\r \u00a0provoked\t\r \u00a0additional\t\r \u00a0MS\t\r \u00a0methods\t\r \u00a0specific\t\r \u00a0for\t\r \u00a0phosphoproteomics,\t\r \u00a0and\t\r \u00a0several\t\r \u00a0databases\t\r \u00a0now\t\r \u00a0exist\t\r \u00a0for\t\r \u00a0documenting\t\r \u00a0the\t\r \u00a0thousands\t\r \u00a0of\t\r \u00a0novel\t\r \u00a0phosphorylation\t\r \u00a0sites\t\r \u00a0emerging\t\r \u00a0from\t\r \u00a0phosphoproteomics\t\r \u00a0data.\t\r \u00a0\t\r \u00a0 1.3.2\t\r \u00a0Lysis\t\r \u00a0methods\t\r \u00a0and\t\r \u00a0low\t\r \u00a0stoichiometry\t\r \u00a0of\t\r \u00a0phosphorylated\t\r \u00a0proteins\t\r \u00a0Although\t\r \u00a0our\t\r \u00a0understanding\t\r \u00a0of\t\r \u00a0phosphatases\t\r \u00a0lags\t\r \u00a0considerably\t\r \u00a0that\t\r \u00a0of\t\r \u00a0kinases,\t\r \u00a0they\t\r \u00a0are\t\r \u00a0thought\t\r \u00a0to\t\r \u00a0offer\t\r \u00a0much\t\r \u00a0less\t\r \u00a0substrate\t\r \u00a0specificity.\t\r \u00a0Many\t\r \u00a0more\t\r \u00a0serine\/threonine\t\r \u00a0kinases\t\r \u00a0exist\t\r \u00a0than\t\r \u00a0cognate\t\r \u00a0phosphatases\t\r \u00a0in\t\r \u00a0the\t\r \u00a0human\t\r \u00a0genome,\t\r \u00a0and,\t\r \u00a0while\t\r \u00a0kinase-\u00ad\u2010substrate\t\r \u00a0specificity\t\r \u00a0is\t\r \u00a0determined\t\r \u00a0by\t\r \u00a0the\t\r \u00a0amino\t\r \u00a0acid\t\r \u00a0sequence\t\r \u00a0surrounding\t\r \u00a0the\t\r \u00a0phosphorylated\t\r \u00a0residue,\t\r \u00a0phosphatases\t\r \u00a0are\t\r \u00a0thought\t\r \u00a0to\t\r \u00a0rely\t\r \u00a0on\t\r \u00a0additional\t\r \u00a0substrate\t\r \u00a0targeting\t\r \u00a0domains(385,\t\r \u00a0386).\t\r \u00a0Thus,\t\r \u00a0phosphatases\t\r \u00a0liberated\t\r \u00a0upon\t\r \u00a0cell\t\r \u00a0lysis\t\r \u00a0can\t\r \u00a0nonspecifically\t\r \u00a0deplete\t\r \u00a0kinase-\u00ad\u2010modified\t\r \u00a0substrates\t\r \u00a0within\t\r \u00a0a\t\r \u00a0sample.\t\r \u00a0By\t\r \u00a0far\t\r \u00a0the\t\r \u00a0most\t\r \u00a0common\t\r \u00a0means\t\r \u00a0of\t\r \u00a0inhibiting\t\r \u00a0phosphatases\t\r \u00a0within\t\r \u00a0phosphoproteomic\t\r \u00a0samples\t\r \u00a0is\t\r \u00a0through\t\r \u00a0the\t\r \u00a0use\t\r \u00a0of\t\r \u00a0commercially\t\r \u00a0available\t\r \u00a0inhibitors.\t\r \u00a0Vanadium\t\r \u00a0oxides\t\r \u00a0such\t\r \u00a0as\t\r \u00a0pervanadate\t\r \u00a0and\t\r \u00a0orthovanadate\t\r \u00a0are\t\r \u00a0used\t\r \u00a0to\t\r \u00a0inhibit\t\r \u00a0all\t\r \u00a0PTPs.\t\r \u00a0However,\t\r \u00a0approximately\t\r \u00a020\t\r \u00a0families\t\r \u00a0of\t\r \u00a0serine\/threonine\t\r \u00a0phosphatases\t\r \u00a0have\t\r \u00a0been\t\r \u00a0classified,\t\r \u00a0the\t\r \u00a0most\t\r \u00a0common\t\r \u00a0of\t\r \u00a0which\t\r \u00a0are\t\r \u00a0protein\t\r \u00a0phosphatase\t\r \u00a01\t\r \u00a0(PP1),\t\r \u00a0PP2A\t\r \u00a0 \t\r \u00a0\t\r \u00a0 45\t\r \u00a0 and\t\r \u00a0PP2B.\t\r \u00a0Inhibitors\t\r \u00a0such\t\r \u00a0as\t\r \u00a0calyculin\t\r \u00a0A\t\r \u00a0are\t\r \u00a0commonly\t\r \u00a0used\t\r \u00a0to\t\r \u00a0inhibit\t\r \u00a0PP1\t\r \u00a0and\t\r \u00a0PP2A,\t\r \u00a0while\t\r \u00a0deltamethrin\t\r \u00a0has\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0inhibit\t\r \u00a0PP2B.\t\r \u00a0However,\t\r \u00a0in\t\r \u00a0a\t\r \u00a0study\t\r \u00a0treating\t\r \u00a0cells\t\r \u00a0with\t\r \u00a0pervanadate,\t\r \u00a0calyculin\t\r \u00a0A,\t\r \u00a0and\t\r \u00a0deltamethrin,\t\r \u00a0and\t\r \u00a0comparing\t\r \u00a0the\t\r \u00a0levels\t\r \u00a0of\t\r \u00a0phosphorylated\t\r \u00a0peptides\t\r \u00a0to\t\r \u00a0an\t\r \u00a0untreated\t\r \u00a0sample,\t\r \u00a0less\t\r \u00a0than\t\r \u00a0one\t\r \u00a0third\t\r \u00a0of\t\r \u00a0the\t\r \u00a0peptides\t\r \u00a0were\t\r \u00a0found\t\r \u00a0to\t\r \u00a0increase\t\r \u00a0more\t\r \u00a0than\t\r \u00a0two\t\r \u00a0fold\t\r \u00a0following\t\r \u00a0treatment,\t\r \u00a0suggesting\t\r \u00a0that\t\r \u00a0several\t\r \u00a0phosphatases\t\r \u00a0are\t\r \u00a0not\t\r \u00a0affected\t\r \u00a0by\t\r \u00a0the\t\r \u00a0inhibitors(387).\t\r \u00a0Similarly,\t\r \u00a0pre-\u00ad\u2010treatment\t\r \u00a0of\t\r \u00a0cells\t\r \u00a0with\t\r \u00a0calyculin\t\r \u00a0A,\t\r \u00a0sodium\t\r \u00a0pervanadate,\t\r \u00a0or\t\r \u00a0two\t\r \u00a0inhibitor\t\r \u00a0cocktails\t\r \u00a0in\t\r \u00a0a\t\r \u00a0separate\t\r \u00a0study\t\r \u00a0resulted\t\r \u00a0in\t\r \u00a0only\t\r \u00a0a\t\r \u00a010-\u00ad\u201040%\t\r \u00a0increase\t\r \u00a0in\t\r \u00a0the\t\r \u00a0number\t\r \u00a0of\t\r \u00a0phosphorylated\t\r \u00a0proteins\t\r \u00a0identified(388).\t\r \u00a0Thus,\t\r \u00a0while\t\r \u00a0current\t\r \u00a0inhibitor-\u00ad\u2010based\t\r \u00a0methods\t\r \u00a0are\t\r \u00a0somewhat\t\r \u00a0effective,\t\r \u00a0alternative\t\r \u00a0techniques\t\r \u00a0such\t\r \u00a0as\t\r \u00a0heat\t\r \u00a0or\t\r \u00a0chaotropic\t\r \u00a0denaturation\t\r \u00a0are\t\r \u00a0needed\t\r \u00a0to\t\r \u00a0completely\t\r \u00a0abolish\t\r \u00a0all\t\r \u00a0phosphatase\t\r \u00a0activity\t\r \u00a0during\t\r \u00a0cell\t\r \u00a0lysis\t\r \u00a0and\t\r \u00a0sample\t\r \u00a0preparation.\t\r \u00a0\t\r \u00a0 1.3.3\t\r \u00a0Phosphopeptide\t\r \u00a0pre-\u00ad\u2010fractionation\t\r \u00a0Due\t\r \u00a0the\t\r \u00a0fact\t\r \u00a0that\t\r \u00a0they\t\r \u00a0are\t\r \u00a0often\t\r \u00a0regulatory\t\r \u00a0and\t\r \u00a0occur\t\r \u00a0with\t\r \u00a0tight\t\r \u00a0spatial\t\r \u00a0and\t\r \u00a0temporal\t\r \u00a0control\t\r \u00a0and\t\r \u00a0at\t\r \u00a0very\t\r \u00a0low\t\r \u00a0stoichiometry,\t\r \u00a0pre-\u00ad\u2010fractionation\t\r \u00a0and\t\r \u00a0enrichment\t\r \u00a0of\t\r \u00a0phosphorylated\t\r \u00a0proteins\t\r \u00a0and\t\r \u00a0peptides\t\r \u00a0is\t\r \u00a0indispensible\t\r \u00a0for\t\r \u00a0large-\u00ad\u2010scale\t\r \u00a0phosphoproteomics(380).\t\r \u00a0Several\t\r \u00a0pre-\u00ad\u2010fractionation\t\r \u00a0methods\t\r \u00a0have\t\r \u00a0been\t\r \u00a0reported\t\r \u00a0for\t\r \u00a0crude\t\r \u00a0purification\t\r \u00a0of\t\r \u00a0phosphorylated\t\r \u00a0proteins\t\r \u00a0and\t\r \u00a0peptides.\t\r \u00a0During\t\r \u00a0one\t\r \u00a0of\t\r \u00a0the\t\r \u00a0first\t\r \u00a0large-\u00ad\u2010scale\t\r \u00a0phosphoproteomic\t\r \u00a0analyses,\t\r \u00a0it\t\r \u00a0was\t\r \u00a0observed\t\r \u00a0that\t\r \u00a0nuclear\t\r \u00a0proteins\t\r \u00a0were\t\r \u00a0over-\u00ad\u2010represented,\t\r \u00a0while\t\r \u00a0mitochondria\t\r \u00a0and\t\r \u00a0mitochondrial\t\r \u00a0proteins\t\r \u00a0were\t\r \u00a0underrepresented\t\r \u00a0in\t\r \u00a0the\t\r \u00a0data,\t\r \u00a0suggesting\t\r \u00a0that\t\r \u00a0subcellular\t\r \u00a0fractionation\t\r \u00a0is\t\r \u00a0beneficial\t\r \u00a0for\t\r \u00a0effective\t\r \u00a0phosphoproteome\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0 \t\r \u00a0\t\r \u00a0 46\t\r \u00a0 individual\t\r \u00a0organelles,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0for\t\r \u00a0globally\t\r \u00a0improving\t\r \u00a0sensitivity\t\r \u00a0in\t\r \u00a0whole\t\r \u00a0cell\t\r \u00a0phosphoproteomic\t\r \u00a0studies(297).\t\r \u00a0At\t\r \u00a0pH\t\r \u00a02.7,\t\r \u00a0only\t\r \u00a0lysine,\t\r \u00a0arginine,\t\r \u00a0histidine,\t\r \u00a0and\t\r \u00a0the\t\r \u00a0amino\t\r \u00a0terminus\t\r \u00a0of\t\r \u00a0a\t\r \u00a0peptide\t\r \u00a0are\t\r \u00a0charged,\t\r \u00a0and\t\r \u00a0the\t\r \u00a0majority\t\r \u00a0of\t\r \u00a0peptides\t\r \u00a0carry\t\r \u00a0a\t\r \u00a0charge\t\r \u00a0of\t\r \u00a02+(389).\t\r \u00a0A\t\r \u00a0negatively\t\r \u00a0charged\t\r \u00a0phosphate\t\r \u00a0moiety\t\r \u00a0reduces\t\r \u00a0this\t\r \u00a0charge\t\r \u00a0state\t\r \u00a0and\t\r \u00a0thus,\t\r \u00a0with\t\r \u00a0increasing\t\r \u00a0salt\t\r \u00a0concentration,\t\r \u00a0phosphopeptides\t\r \u00a0elute\t\r \u00a0earlier\t\r \u00a0than\t\r \u00a0the\t\r \u00a0majority\t\r \u00a0of\t\r \u00a0non-\u00ad\u2010phosphorylated\t\r \u00a0peptides\t\r \u00a0from\t\r \u00a0an\t\r \u00a0SCX\t\r \u00a0column,\t\r \u00a0which\t\r \u00a0has\t\r \u00a0been\t\r \u00a0used\t\r \u00a0extensively\t\r \u00a0to\t\r \u00a0crudely\t\r \u00a0purify\t\r \u00a0phosphorylated\t\r \u00a0peptides(297,\t\r \u00a0389,\t\r \u00a0390).\t\r \u00a0The\t\r \u00a0use\t\r \u00a0of\t\r \u00a0SAX\t\r \u00a0chromatography\t\r \u00a0has\t\r \u00a0also\t\r \u00a0been\t\r \u00a0described(359,\t\r \u00a0391,\t\r \u00a0392).\t\r \u00a0However,\t\r \u00a0acidic\t\r \u00a0peptides\t\r \u00a0also\t\r \u00a0bind\t\r \u00a0strongly\t\r \u00a0to\t\r \u00a0the\t\r \u00a0material\t\r \u00a0and\t\r \u00a0it\t\r \u00a0has\t\r \u00a0been\t\r \u00a0reported\t\r \u00a0that\t\r \u00a0phosphopeptides\t\r \u00a0bind\t\r \u00a0with\t\r \u00a0very\t\r \u00a0strong\t\r \u00a0affinity\t\r \u00a0and\t\r \u00a0can\t\r \u00a0be\t\r \u00a0difficult\t\r \u00a0to\t\r \u00a0remove\t\r \u00a0from\t\r \u00a0the\t\r \u00a0resin(393).\t\r \u00a0\t\r \u00a0During\t\r \u00a0hydrophilic\t\r \u00a0interaction\t\r \u00a0chromatography\t\r \u00a0(HILIC),\t\r \u00a0peptides\t\r \u00a0are\t\r \u00a0applied\t\r \u00a0to\t\r \u00a0a\t\r \u00a0hydrophilic\t\r \u00a0stationary\t\r \u00a0phase\t\r \u00a0in\t\r \u00a0an\t\r \u00a0organic\t\r \u00a0mobile\t\r \u00a0phase\t\r \u00a0and\t\r \u00a0separation\t\r \u00a0is\t\r \u00a0achieved\t\r \u00a0based\t\r \u00a0on\t\r \u00a0polarity(394,\t\r \u00a0395).\t\r \u00a0However,\t\r \u00a0it\t\r \u00a0has\t\r \u00a0been\t\r \u00a0reported\t\r \u00a0to\t\r \u00a0very\t\r \u00a0strongly\t\r \u00a0retain\t\r \u00a0very\t\r \u00a0acidic\t\r \u00a0and\t\r \u00a0basic\t\r \u00a0peptides.\t\r \u00a0Alternatively,\t\r \u00a0electrostatic\t\r \u00a0repulsion-\u00ad\u2010hydrophilic\t\r \u00a0interaction\t\r \u00a0chromatography\t\r \u00a0(ERLIC)\t\r \u00a0employs\t\r \u00a0HILIC\t\r \u00a0on\t\r \u00a0a\t\r \u00a0weak\t\r \u00a0anion-\u00ad\u2010exchange\t\r \u00a0column\t\r \u00a0(WAX)(396).\t\r \u00a0During\t\r \u00a0ERLIC,\t\r \u00a0peptides\t\r \u00a0are\t\r \u00a0run\t\r \u00a0at\t\r \u00a0low\t\r \u00a0pH\t\r \u00a0(~2)\t\r \u00a0in\t\r \u00a0an\t\r \u00a0organic\t\r \u00a0mobile\t\r \u00a0phase\t\r \u00a0and\t\r \u00a0eluted\t\r \u00a0with\t\r \u00a0increasing\t\r \u00a0salt\t\r \u00a0concentration.\t\r \u00a0Phosphopeptides\t\r \u00a0are\t\r \u00a0retained\t\r \u00a0much\t\r \u00a0better\t\r \u00a0than\t\r \u00a0non-\u00ad\u2010phosphorylated\t\r \u00a0peptides\t\r \u00a0on\t\r \u00a0an\t\r \u00a0ERLIC\t\r \u00a0column,\t\r \u00a0and\t\r \u00a0it\t\r \u00a0has\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0be\t\r \u00a0very\t\r \u00a0effective\t\r \u00a0for\t\r \u00a0phosphopeptide\t\r \u00a0pre-\u00ad\u2010fractionation(397).\t\r \u00a0In\t\r \u00a0addition\t\r \u00a0to\t\r \u00a0these\t\r \u00a0subcellular\t\r \u00a0and\t\r \u00a0chromatographic\t\r \u00a0methods,\t\r \u00a0precipitation\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0by\t\r \u00a0co-\u00ad\u2010precipitation\t\r \u00a0with\t\r \u00a0calcium\t\r \u00a0phosphate,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0 \t\r \u00a0\t\r \u00a0 47\t\r \u00a0 precipitation\t\r \u00a0in\t\r \u00a0barium\t\r \u00a0and\t\r \u00a0acetone\t\r \u00a0has\t\r \u00a0been\t\r \u00a0reported(398,\t\r \u00a0399).\t\r \u00a0The\t\r \u00a0acidic\t\r \u00a0nature\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0also\t\r \u00a0enables\t\r \u00a0their\t\r \u00a0efficient\t\r \u00a0separation\t\r \u00a0by\t\r \u00a0IEF,\t\r \u00a0where\t\r \u00a0methyl-\u00ad\u2010esterification\t\r \u00a0of\t\r \u00a0carboxyl\t\r \u00a0groups\t\r \u00a0has\t\r \u00a0been\t\r \u00a0employed\t\r \u00a0to\t\r \u00a0reduce\t\r \u00a0co-\u00ad\u2010migration\t\r \u00a0of\t\r \u00a0acidic\t\r \u00a0peptides(400-\u00ad\u2010402).\t\r \u00a0\t\r \u00a0 1.3.4\t\r \u00a0Phosphopeptide\t\r \u00a0enrichment\t\r \u00a0The\t\r \u00a0methods\t\r \u00a0described\t\r \u00a0above\t\r \u00a0offer\t\r \u00a0crude\t\r \u00a0phosphopeptide\t\r \u00a0purification,\t\r \u00a0and\t\r \u00a0in\t\r \u00a0some\t\r \u00a0cases\t\r \u00a0effective\t\r \u00a0fractionation\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0themselves.\t\r \u00a0However,\t\r \u00a0due\t\r \u00a0to\t\r \u00a0their\t\r \u00a0very\t\r \u00a0low\t\r \u00a0abundance,\t\r \u00a0additional\t\r \u00a0techniques\t\r \u00a0offering\t\r \u00a0very\t\r \u00a0high\t\r \u00a0selectivity\t\r \u00a0for\t\r \u00a0phosphate\t\r \u00a0are\t\r \u00a0often\t\r \u00a0preferred\t\r \u00a0or\t\r \u00a0used\t\r \u00a0in\t\r \u00a0combination\t\r \u00a0and\t\r \u00a0downstream\t\r \u00a0of\t\r \u00a0these\t\r \u00a0techniques.\t\r \u00a0For\t\r \u00a0highly\t\r \u00a0selective\t\r \u00a0enrichment\t\r \u00a0of\t\r \u00a0tyrosine\t\r \u00a0phosphorylated\t\r \u00a0peptides\t\r \u00a0and\t\r \u00a0proteins,\t\r \u00a0immunoprecipitation\t\r \u00a0using\t\r \u00a0phosphotyrosine\t\r \u00a0(pY)\t\r \u00a0specific\t\r \u00a0antibodies\t\r \u00a0has\t\r \u00a0proven\t\r \u00a0to\t\r \u00a0be\t\r \u00a0very\t\r \u00a0effective(403,\t\r \u00a0404).\t\r \u00a0However,\t\r \u00a0with\t\r \u00a0only\t\r \u00a0a\t\r \u00a0few\t\r \u00a0exceptions,\t\r \u00a0poor\t\r \u00a0specificity\t\r \u00a0has\t\r \u00a0been\t\r \u00a0observed\t\r \u00a0for\t\r \u00a0pan-\u00ad\u2010phosphoserine\t\r \u00a0(pS)\t\r \u00a0and\t\r \u00a0\u2013phosphothreonine\t\r \u00a0(pT)\t\r \u00a0antibodies(405).\t\r \u00a0This,\t\r \u00a0combined\t\r \u00a0with\t\r \u00a0the\t\r \u00a0fact\t\r \u00a0that\t\r \u00a0only\t\r \u00a0~2%\t\r \u00a0of\t\r \u00a0eukaryotic\t\r \u00a0phosphorylation\t\r \u00a0sites\t\r \u00a0have\t\r \u00a0been\t\r \u00a0estimated\t\r \u00a0to\t\r \u00a0occur\t\r \u00a0on\t\r \u00a0tyrosine\t\r \u00a0residues,\t\r \u00a0suggests\t\r \u00a0that\t\r \u00a0additional\t\r \u00a0purification\t\r \u00a0techniques\t\r \u00a0are\t\r \u00a0required\t\r \u00a0for\t\r \u00a0truly\t\r \u00a0global\t\r \u00a0analyses(297).\t\r \u00a0\t\r \u00a0Immobilized\t\r \u00a0metal\t\r \u00a0affinity\t\r \u00a0chromatography\t\r \u00a0(IMAC)\t\r \u00a0and\t\r \u00a0metal\t\r \u00a0oxide\t\r \u00a0chromatography\t\r \u00a0(MOC)\t\r \u00a0represent\t\r \u00a0two\t\r \u00a0widely\t\r \u00a0used\t\r \u00a0methods\t\r \u00a0for\t\r \u00a0the\t\r \u00a0selective\t\r \u00a0enrichment\t\r \u00a0of\t\r \u00a0phosphoserine\t\r \u00a0-\u00ad\u2010threonine\t\r \u00a0and\t\r \u00a0\u2013tyrosine\t\r \u00a0containing\t\r \u00a0peptides.\t\r \u00a0IMAC\t\r \u00a0is\t\r \u00a0based\t\r \u00a0on\t\r \u00a0the\t\r \u00a0high\t\r \u00a0affinity\t\r \u00a0of\t\r \u00a0phosphate\t\r \u00a0groups\t\r \u00a0for\t\r \u00a0metal\t\r \u00a0ions\t\r \u00a0such\t\r \u00a0as\t\r \u00a0Fe3+,\t\r \u00a0Zn2+,\t\r \u00a0and\t\r \u00a0Ga3+(406,\t\r \u00a0407)\t\r \u00a0(Figure\t\r \u00a01.4A).\t\r \u00a0One\t\r \u00a0of\t\r \u00a0the\t\r \u00a0main\t\r \u00a0limitations\t\r \u00a0of\t\r \u00a0IMAC\t\r \u00a0involves\t\r \u00a0the\t\r \u00a0 \t\r \u00a0\t\r \u00a0 48\t\r \u00a0 nonspecific\t\r \u00a0retention\t\r \u00a0of\t\r \u00a0generally\t\r \u00a0acidic\t\r \u00a0peptides\t\r \u00a0due\t\r \u00a0to\t\r \u00a0the\t\r \u00a0affinity\t\r \u00a0of\t\r \u00a0negatively\t\r \u00a0charged\t\r \u00a0carboxylates\t\r \u00a0for\t\r \u00a0the\t\r \u00a0positively\t\r \u00a0charged\t\r \u00a0metal\t\r \u00a0ions\t\r \u00a0in\t\r \u00a0the\t\r \u00a0matrix\t\r \u00a0(Figure\t\r \u00a01.4C).\t\r \u00a0Derivatizing\t\r \u00a0carboxylic\t\r \u00a0acid\t\r \u00a0moieties\t\r \u00a0to\t\r \u00a0methyl\t\r \u00a0esters\t\r \u00a0has\t\r \u00a0been\t\r \u00a0reported\t\r \u00a0to\t\r \u00a0greatly\t\r \u00a0reduce\t\r \u00a0contamination\t\r \u00a0by\t\r \u00a0acidic\t\r \u00a0peptides.\t\r \u00a0However\t\r \u00a0this\t\r \u00a0also\t\r \u00a0facilitates\t\r \u00a0side\t\r \u00a0reactions,\t\r \u00a0which\t\r \u00a0complicate\t\r \u00a0analysis\t\r \u00a0and\t\r \u00a0can\t\r \u00a0cause\t\r \u00a0sample\t\r \u00a0loss(375,\t\r \u00a0408-\u00ad\u2010410).\t\r \u00a0Alternatively,\t\r \u00a0MOC\t\r \u00a0utilizes\t\r \u00a0the\t\r \u00a0affinity\t\r \u00a0of\t\r \u00a0acidified\t\r \u00a0phosphoric\t\r \u00a0acid\t\r \u00a0for\t\r \u00a0metal\t\r \u00a0oxides\t\r \u00a0such\t\r \u00a0as\t\r \u00a0TiO2\t\r \u00a0and\t\r \u00a0ZrO2(411)\t\r \u00a0(Figure\t\r \u00a01.4A).\t\r \u00a0Without\t\r \u00a0requiring\t\r \u00a0chemical\t\r \u00a0modification\t\r \u00a0of\t\r \u00a0carboxylates,\t\r \u00a0MOC\t\r \u00a0makes\t\r \u00a0use\t\r \u00a0of\t\r \u00a0dihydroxy-\u00ad\u2010benzoic\t\r \u00a0acid\t\r \u00a0(DHB),\t\r \u00a0or\t\r \u00a0aliphatic\t\r \u00a0hydroxyl\t\r \u00a0acids\t\r \u00a0(i.e.,\t\r \u00a0lactic\t\r \u00a0or\t\r \u00a0\u03b2-\u00ad\u2010hydroxypropanoic\t\r \u00a0acid)\t\r \u00a0to\t\r \u00a0compete\t\r \u00a0off\t\r \u00a0acidic\t\r \u00a0but\t\r \u00a0not\t\r \u00a0phosphorylated\t\r \u00a0peptides\t\r \u00a0from\t\r \u00a0the\t\r \u00a0matrix\t\r \u00a0(Figure\t\r \u00a01.4B)\t\r \u00a0and\t\r \u00a0has\t\r \u00a0been\t\r \u00a0reported\t\r \u00a0to\t\r \u00a0yield\t\r \u00a0a\t\r \u00a0far\t\r \u00a0higher\t\r \u00a0selectivity\t\r \u00a0towards\t\r \u00a0phosphorylated\t\r \u00a0peptides\t\r \u00a0versus\t\r \u00a0carboxylates\t\r \u00a0than\t\r \u00a0does\t\r \u00a0IMAC(376,\t\r \u00a0412-\u00ad\u2010414).\t\r \u00a0A\t\r \u00a0combined\t\r \u00a0approach\t\r \u00a0termed\t\r \u00a0Sequential\t\r \u00a0Elution\t\r \u00a0from\t\r \u00a0IMAC\t\r \u00a0(SIMAC)\t\r \u00a0has\t\r \u00a0also\t\r \u00a0been\t\r \u00a0developed,\t\r \u00a0which\t\r \u00a0first\t\r \u00a0elutes\t\r \u00a0monophosphorylated\t\r \u00a0and\t\r \u00a0acidic\t\r \u00a0peptides\t\r \u00a0from\t\r \u00a0an\t\r \u00a0IMAC\t\r \u00a0column\t\r \u00a0under\t\r \u00a0acidic\t\r \u00a0conditions,\t\r \u00a0followed\t\r \u00a0by\t\r \u00a0the\t\r \u00a0elution\t\r \u00a0of\t\r \u00a0polyphosphorylated\t\r \u00a0peptides\t\r \u00a0under\t\r \u00a0basic\t\r \u00a0conditions(415).\t\r \u00a0The\t\r \u00a0monophosphorylated\t\r \u00a0peptides\t\r \u00a0are\t\r \u00a0then\t\r \u00a0separated\t\r \u00a0from\t\r \u00a0acidic\t\r \u00a0peptides\t\r \u00a0using\t\r \u00a0TiO2\t\r \u00a0chromatography\t\r \u00a0and\t\r \u00a0the\t\r \u00a0two\t\r \u00a0eluates\t\r \u00a0are\t\r \u00a0analyzed\t\r \u00a0separately\t\r \u00a0by\t\r \u00a0LC-\u00ad\u2010MSn.\t\r \u00a0SIMAC\t\r \u00a0has\t\r \u00a0been\t\r \u00a0reported\t\r \u00a0to\t\r \u00a0more\t\r \u00a0than\t\r \u00a0double\t\r \u00a0the\t\r \u00a0number\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0identified\t\r \u00a0by\t\r \u00a0TiO2\t\r \u00a0alone.\t\r \u00a0 \t\r \u00a0\t\r \u00a0 49\t\r \u00a0 Figure\t\r \u00a01.4\t\r \u00a0Diagram\t\r \u00a0of\t\r \u00a0IMAC\t\r \u00a0and\t\r \u00a0TiO2\t\r \u00a0phosphopeptide\t\r \u00a0enrichment\t\r \u00a0methods\t\r \u00a0 \t\r \u00a0A)\t\r \u00a0The\t\r \u00a0co-\u00ad\u2010ordination\t\r \u00a0of\t\r \u00a0IMAC\t\r \u00a0and\t\r \u00a0TiO2\t\r \u00a0resin\t\r \u00a0with\t\r \u00a0a\t\r \u00a0phosphorylated\t\r \u00a0peptide\t\r \u00a0is\t\r \u00a0shown.\t\r \u00a0B)\t\r \u00a0The\t\r \u00a0co-\u00ad\u2010ordination\t\r \u00a0of\t\r \u00a0TiO2\t\r \u00a0resin\t\r \u00a0with\t\r \u00a0DHB\t\r \u00a0(middle)\t\r \u00a0and\t\r \u00a0lactic\t\r \u00a0acid\t\r \u00a0(right)\t\r \u00a0is\t\r \u00a0shown.\t\r \u00a0C)\t\r \u00a0The\t\r \u00a0co-\u00ad\u2010ordination\t\r \u00a0of\t\r \u00a0IMAC\t\r \u00a0and\t\r \u00a0TiO2\t\r \u00a0with\t\r \u00a0a\t\r \u00a0peptide\t\r \u00a0containing\t\r \u00a0an\t\r \u00a0acidic\t\r \u00a0residue\t\r \u00a0(carboxyl\t\r \u00a0group)\t\r \u00a0is\t\r \u00a0shown.\t\r \u00a0Peptides\t\r \u00a0are\t\r \u00a0represented\t\r \u00a0by\t\r \u00a0a\t\r \u00a0red\t\r \u00a0wave\t\r \u00a0line.\t\r \u00a0\t\r \u00a0 The\t\r \u00a0focus\t\r \u00a0here\t\r \u00a0has\t\r \u00a0been\t\r \u00a0primarily\t\r \u00a0on\t\r \u00a0phosphopeptide\t\r \u00a0enrichment\t\r \u00a0techniques\t\r \u00a0that\t\r \u00a0do\t\r \u00a0not\t\r \u00a0require\t\r \u00a0chemical\t\r \u00a0derivatization\t\r \u00a0of\t\r \u00a0the\t\r \u00a0phosphate\t\r \u00a0moiety\t\r \u00a0itself.\t\r \u00a0While\t\r \u00a0several\t\r \u00a0derivatization\t\r \u00a0methods,\t\r \u00a0e.g.,\t\r \u00a0\u03b2-\u00ad\u2010elimination,\t\r \u00a0have\t\r \u00a0been\t\r \u00a0applied\t\r \u00a0to\t\r \u00a0phosphoproteomic\t\r \u00a0studies\t\r \u00a0and\t\r 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\u00a0418).\t\r \u00a0\t\r \u00a0 \t\r \u00a0\t\r \u00a0 50\t\r \u00a0 1.3.5\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0methods\t\r \u00a0for\t\r \u00a0phosphoproteomics\t\r \u00a0It\t\r \u00a0has\t\r \u00a0been\t\r \u00a0believed\t\r \u00a0for\t\r \u00a0several\t\r \u00a0years\t\r \u00a0that\t\r \u00a0CID\t\r \u00a0is\t\r \u00a0ineffective\t\r \u00a0for\t\r \u00a0phosphopeptide\t\r \u00a0analysis\t\r \u00a0as\t\r \u00a0neutral\t\r \u00a0loss\t\r \u00a0of\t\r \u00a0phosphoric\t\r \u00a0acid\t\r \u00a0(H3PO4,\t\r \u00a0from\t\r \u00a0pS\t\r \u00a0and\t\r \u00a0pT)\t\r \u00a0would\t\r \u00a0occur\t\r \u00a0before\t\r \u00a0backbone\t\r \u00a0cleavage,\t\r \u00a0giving\t\r \u00a0insufficient\t\r \u00a0backbone\t\r \u00a0fragmentation\t\r \u00a0for\t\r \u00a0effective\t\r \u00a0identification\t\r \u00a0of\t\r \u00a0the\t\r \u00a0peptide(419)\t\r \u00a0(Figure\t\r \u00a01.5A).\t\r \u00a0Thus,\t\r \u00a0methods\t\r \u00a0such\t\r \u00a0as\t\r \u00a0MS3\t\r \u00a0and\t\r \u00a0MultiStage\t\r \u00a0Activation\t\r \u00a0have\t\r \u00a0been\t\r \u00a0employed\t\r \u00a0to\t\r \u00a0impose\t\r \u00a0additional\t\r \u00a0activation\t\r \u00a0events\t\r \u00a0on\t\r \u00a0preselected\t\r \u00a0neutral\t\r \u00a0loss\t\r \u00a0peaks(389,\t\r \u00a0420).\t\r \u00a0They\t\r \u00a0differ\t\r \u00a0in\t\r \u00a0the\t\r \u00a0sense\t\r \u00a0that\t\r \u00a0during\t\r \u00a0MS3\t\r \u00a0the\t\r \u00a0peak\t\r \u00a0selected\t\r \u00a0for\t\r \u00a0additional\t\r \u00a0fragmentation\t\r \u00a0is\t\r \u00a0isolated\t\r \u00a0prior\t\r \u00a0to\t\r \u00a0activation\t\r \u00a0and\t\r \u00a0results\t\r \u00a0in\t\r \u00a0an\t\r \u00a0entirely\t\r \u00a0new\t\r \u00a0set\t\r \u00a0of\t\r \u00a0product\t\r \u00a0ions\t\r \u00a0(Figure\t\r \u00a01.5B),\t\r \u00a0while\t\r \u00a0during\t\r \u00a0MultiStage\t\r \u00a0Activation\t\r \u00a0the\t\r \u00a0second\t\r \u00a0isolation\t\r \u00a0step\t\r \u00a0is\t\r \u00a0eliminated\t\r \u00a0and\t\r \u00a0the\t\r \u00a0spectra\t\r \u00a0contain\t\r \u00a0product\t\r \u00a0ions\t\r \u00a0from\t\r \u00a0both\t\r \u00a0activation\t\r \u00a0events,\t\r \u00a0making\t\r \u00a0it\t\r \u00a0a\t\r \u00a0pseudo-\u00ad\u2010MS3\t\r \u00a0approach\t\r \u00a0(Figure\t\r \u00a01.5C).\t\r \u00a0MultiStage\t\r \u00a0Activation\t\r \u00a0also\t\r \u00a0enables\t\r \u00a0higher\t\r \u00a0MSn\t\r \u00a0activations\t\r \u00a0in\t\r \u00a0cases\t\r \u00a0where\t\r \u00a0multiply\t\r \u00a0phosphorylated\t\r \u00a0peptides\t\r \u00a0undergo\t\r \u00a0sequential\t\r \u00a0neutral\t\r \u00a0losses.\t\r \u00a0Following\t\r \u00a0MS3\t\r \u00a0or\t\r \u00a0MultiStage\t\r \u00a0Activation,\t\r \u00a0pS\t\r \u00a0and\t\r \u00a0pT\t\r \u00a0sites\t\r \u00a0are\t\r \u00a0recognized\t\r \u00a0by\t\r \u00a0the\t\r \u00a0presence\t\r \u00a0of\t\r \u00a0fragment\t\r \u00a0ions\t\r \u00a0containing\t\r \u00a0dehydroalanine\t\r \u00a0and\t\r \u00a0dehydrobutyric\t\r \u00a0acid\t\r \u00a0respectively,\t\r \u00a0while\t\r \u00a0phosphotyrosine\t\r \u00a0residues\t\r \u00a0are\t\r \u00a0rarely\t\r \u00a0observed\t\r \u00a0to\t\r \u00a0undergo\t\r \u00a0the\t\r \u00a0described\t\r \u00a0neutral\t\r \u00a0loss(421).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 51\t\r \u00a0 Figure\t\r \u00a01.5\t\r \u00a0Schematic\t\r \u00a0of\t\r \u00a0MS2,\t\r \u00a0MS3\t\r \u00a0and\t\r \u00a0MultiStage\t\r \u00a0Activation\t\r \u00a0for\t\r \u00a0phosphoproteomics\t\r \u00a0 \t\r \u00a0A)\t\r \u00a0During\t\r \u00a0MS2\t\r \u00a0a\t\r \u00a0phosphopeptide\t\r \u00a0is\t\r \u00a0selected\t\r \u00a0for\t\r \u00a0CID\t\r \u00a0and\t\r \u00a0isolated\t\r \u00a0(1a).\t\r \u00a0The\t\r \u00a0resulting\t\r \u00a0fragments\t\r \u00a0consist\t\r \u00a0either\t\r \u00a0of\t\r \u00a0primarily\t\r \u00a0y-\u00ad\u2010\t\r \u00a0and\t\r \u00a0b-\u00ad\u2010ions\t\r \u00a0(2a)\t\r \u00a0or\t\r \u00a0a\t\r \u00a0dominant\t\r \u00a0neutral\t\r \u00a0loss\t\r \u00a0peak\t\r \u00a0corresponding\t\r \u00a0to\t\r \u00a0the\t\r \u00a0loss\t\r \u00a0of\t\r \u00a0phosphoric\t\r \u00a0acid\t\r \u00a0(-\u00ad\u201098Da)\t\r \u00a0(3a).\t\r \u00a0B)\t\r \u00a0During\t\r \u00a0MS3\t\r \u00a0a\t\r \u00a0phosphopeptide\t\r \u00a0is\t\r \u00a0selected\t\r \u00a0for\t\r \u00a0CID\t\r \u00a0and\t\r \u00a0isolated\t\r \u00a0(1b).\t\r \u00a0If\t\r \u00a0a\t\r \u00a0dominant\t\r \u00a0neutral\t\r \u00a0loss\t\r \u00a0peak\t\r \u00a0is\t\r \u00a0detected\t\r \u00a0(-\u00ad\u201098Da)\t\r \u00a0(2b),\t\r \u00a0the\t\r \u00a0neutral\t\r \u00a0loss\t\r \u00a0peak\t\r \u00a0is\t\r \u00a0re-\u00ad\u2010isolated\t\r \u00a0(3b)\t\r \u00a0and\t\r \u00a0an\t\r \u00a0additional\t\r \u00a0round\t\r \u00a0of\t\r \u00a0CID\t\r \u00a0yields\t\r \u00a0fragment\t\r \u00a0ions\t\r \u00a0from\t\r \u00a0the\t\r \u00a0neutral\t\r \u00a0loss\t\r \u00a0peak\t\r \u00a0(4b).\t\r \u00a0C)\t\r \u00a0During\t\r \u00a0MultiStage\t\r \u00a0Activation\t\r \u00a0a\t\r \u00a0peptide\t\r \u00a0is\t\r \u00a0selected\t\r \u00a0for\t\r \u00a0CID\t\r \u00a0and\t\r \u00a0isolated\t\r \u00a0(1c).\t\r \u00a0If\t\r \u00a0a\t\r \u00a0dominant\t\r \u00a0neutral\t\r \u00a0loss\t\r \u00a0peak\t\r \u00a0is\t\r \u00a0detected\t\r \u00a0(-\u00ad\u201098Da)\t\r \u00a0(2c)\t\r \u00a0the\t\r \u00a0neutral\t\r \u00a0loss\t\r \u00a0peak\t\r \u00a0is\t\r \u00a0re-\u00ad\u2010activated\t\r \u00a0by\t\r \u00a0CID\t\r \u00a0(2c)\t\r \u00a0yielding\t\r \u00a0a\t\r \u00a0product\t\r \u00a0ion\t\r \u00a0spectra\t\r \u00a0containing\t\r \u00a0fragment\t\r \u00a0ions\t\r \u00a0from\t\r \u00a0both\t\r \u00a0collision\t\r \u00a0events.\t\r \u00a0Red\t\r \u00a0and\t\r \u00a0blue\t\r \u00a0bars\t\r \u00a0represent\t\r \u00a0parent\t\r \u00a0ions\t\r \u00a0and\t\r \u00a0fragment\t\r \u00a0ions\t\r \u00a0from\t\r \u00a0the\t\r \u00a0first\t\r \u00a0and\t\r \u00a0second\t\r \u00a0fragmentation\t\r \u00a0events\t\r \u00a0respectively.\t\r \u00a0\t\r \u00a0 However,\t\r \u00a0the\t\r \u00a0advantages\t\r \u00a0of\t\r \u00a0using\t\r \u00a0neutral\t\r \u00a0loss-\u00ad\u2010driven\t\r \u00a0MS3\t\r \u00a0and\t\r 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\u00a0intensity\t\r \u00a0of\t\r \u00a0MS2\t\r \u00a0spectra,\t\r \u00a0and\t\r \u00a0rarely\t\r \u00a0produce\t\r \u00a0more\t\r \u00a0informative\t\r \u00a0ions\t\r \u00a0than\t\r \u00a0normal\t\r \u00a0MS2\t\r \u00a0CID\t\r \u00a0when\t\r \u00a0high\t\r \u00a0mass-\u00ad\u2010accuracy\t\r \u00a0instruments\t\r \u00a0are\t\r \u00a0used.\t\r \u00a0However,\t\r \u00a0in\t\r \u00a0a\t\r \u00a0subsequent\t\r \u00a0study,\t\r \u00a0both\t\r \u00a0 \t\r \u00a0\t\r \u00a0 52\t\r \u00a0 MultiStage\t\r \u00a0Activation\t\r \u00a0and\t\r \u00a0MS2\t\r \u00a0were\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0outperform\t\r \u00a0MS3\t\r \u00a0methodologies.\t\r \u00a0Here,\t\r \u00a0MultiStage\t\r \u00a0Activation\t\r \u00a0resulted\t\r \u00a0in\t\r \u00a0a\t\r \u00a06%\t\r \u00a0increase\t\r \u00a0in\t\r \u00a0the\t\r \u00a0number\t\r \u00a0of\t\r \u00a0unique\t\r \u00a0phosphopeptides\t\r \u00a0identified,\t\r \u00a0and\t\r \u00a0all\t\r \u00a0three\t\r \u00a0methods\t\r \u00a0performed\t\r \u00a0equally\t\r \u00a0well\t\r \u00a0in\t\r \u00a0localizing\t\r \u00a0phosphorylation\t\r \u00a0sites(423).\t\r \u00a0In\t\r \u00a0addition,\t\r \u00a0while\t\r \u00a0pseudo-\u00ad\u2010MS3\t\r \u00a0scans\t\r \u00a0may\t\r \u00a0generate\t\r \u00a0slightly\t\r \u00a0richer\t\r \u00a0spectra\t\r \u00a0than\t\r \u00a0MS2\t\r \u00a0scans,\t\r \u00a0they\t\r \u00a0can\t\r \u00a0also\t\r \u00a0complicate\t\r \u00a0analysis\t\r \u00a0and\t\r \u00a0cause\t\r \u00a0ambiguity\t\r \u00a0in\t\r \u00a0phosphosite\t\r \u00a0localization.\t\r \u00a0For\t\r \u00a0example,\t\r \u00a0the\t\r \u00a0neutral\t\r \u00a0loss\t\r \u00a0of\t\r \u00a0methanesulfenic\t\r \u00a0acid\t\r \u00a0from\t\r \u00a0methionine\t\r \u00a0and\t\r \u00a0phosphoric\t\r \u00a0acid\t\r \u00a0from\t\r \u00a0threonine,\t\r \u00a0both\t\r \u00a0yield\t\r \u00a0dehydrobutyric\t\r \u00a0acid.\t\r \u00a0This\t\r \u00a0can\t\r \u00a0impose\t\r \u00a0false\t\r \u00a0positives\t\r \u00a0from\t\r \u00a0MS3\t\r \u00a0scans\t\r \u00a0where\t\r \u00a0fragment\t\r \u00a0ions\t\r \u00a0containing\t\r \u00a0dehydrobutyric\t\r \u00a0acid\t\r \u00a0are\t\r \u00a0used\t\r \u00a0to\t\r \u00a0localize\t\r \u00a0pT\t\r \u00a0sites\t\r \u00a0in\t\r \u00a0instances\t\r \u00a0of\t\r \u00a0methionine\t\r \u00a0and\t\r \u00a0threonine\t\r \u00a0isomerism(424).\t\r \u00a0Similarly,\t\r \u00a0it\t\r \u00a0has\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0that,\t\r \u00a0contrary\t\r \u00a0to\t\r \u00a0the\t\r \u00a0\u03b2-\u00ad\u2010elimination\t\r \u00a0reaction\t\r \u00a0that\t\r \u00a0was\t\r \u00a0believed\t\r \u00a0to\t\r \u00a0explain\t\r \u00a0the\t\r \u00a0neutral\t\r \u00a0loss\t\r \u00a0of\t\r \u00a0phosphoric\t\r \u00a0acid\t\r \u00a0from\t\r \u00a0pS\t\r \u00a0and\t\r \u00a0pT-\u00ad\u2010containing\t\r \u00a0peptides,\t\r \u00a0the\t\r \u00a0neutral\t\r \u00a0loss\t\r \u00a0occurs\t\r \u00a0through\t\r \u00a0a\t\r \u00a0SN2\t\r \u00a0charge-\u00ad\u2010directed\t\r \u00a0mechanism\t\r \u00a0that\t\r \u00a0can\t\r \u00a0result\t\r \u00a0in\t\r \u00a0the\t\r \u00a0sequential\t\r \u00a0neutral\t\r \u00a0loss\t\r \u00a0of\t\r \u00a0metaphosphoric\t\r \u00a0acid\t\r \u00a0and\t\r \u00a0water\t\r \u00a0from\t\r \u00a0two\t\r \u00a0different\t\r \u00a0residues\t\r \u00a0in\t\r \u00a0a\t\r \u00a0peptide(425).\t\r \u00a0In\t\r \u00a0this\t\r \u00a0case,\t\r \u00a0localizing\t\r \u00a0a\t\r \u00a0phosphorylation\t\r \u00a0site\t\r \u00a0based\t\r \u00a0on\t\r \u00a0the\t\r \u00a0presence\t\r \u00a0of\t\r \u00a0dehydroalanine\t\r \u00a0or\t\r \u00a0dehydrobutyric\t\r \u00a0acid\t\r \u00a0may\t\r \u00a0mislocalize\t\r \u00a0the\t\r \u00a0site.\t\r \u00a0During\t\r \u00a0the\t\r \u00a0relatively\t\r \u00a0long\t\r \u00a0activation\t\r \u00a0times\t\r \u00a0required\t\r \u00a0for\t\r \u00a0CID\t\r \u00a0in\t\r \u00a0ion\t\r \u00a0trap\t\r \u00a0mass\t\r \u00a0spectrometers,\t\r \u00a0almost\t\r \u00a0half\t\r \u00a0of\t\r \u00a0the\t\r \u00a0analyzed\t\r \u00a0peptides\t\r \u00a0can\t\r \u00a0transfer\t\r \u00a0a\t\r \u00a0phosphate\t\r \u00a0group\t\r \u00a0to\t\r \u00a0a\t\r \u00a0previously\t\r \u00a0unmodified\t\r \u00a0site\t\r \u00a0during\t\r \u00a0activation.\t\r \u00a0This\t\r \u00a0increases\t\r \u00a0with\t\r \u00a0proton\t\r \u00a0mobility\t\r \u00a0and\t\r \u00a0at\t\r \u00a0lower\t\r \u00a0charge\t\r \u00a0states,\t\r \u00a0and\t\r \u00a0is\t\r \u00a0dependent\t\r \u00a0on\t\r \u00a0the\t\r \u00a0relatively\t\r \u00a0long\t\r \u00a0activation\t\r \u00a0times\t\r \u00a0employed\t\r \u00a0for\t\r \u00a0CID\t\r \u00a0in\t\r \u00a0an\t\r \u00a0ion\t\r \u00a0trap.\t\r \u00a0Thus\t\r \u00a0to\t\r \u00a0minimize\t\r \u00a0such\t\r \u00a0artifacts,\t\r \u00a0peptides\t\r \u00a0should\t\r \u00a0be\t\r \u00a0activated\t\r \u00a0in\t\r \u00a0either\t\r \u00a0a\t\r \u00a0quadrupole,\t\r \u00a0or\t\r \u00a0using\t\r \u00a0alternative\t\r \u00a0fragmentation\t\r \u00a0methods\t\r \u00a0such\t\r \u00a0as\t\r \u00a0electron\t\r \u00a0capture\t\r \u00a0dissociation\t\r \u00a0(ECD)\t\r \u00a0or\t\r \u00a0 \t\r \u00a0\t\r \u00a0 53\t\r \u00a0 ETD,\t\r \u00a0both\t\r \u00a0of\t\r \u00a0which\t\r \u00a0allow\t\r \u00a0fragmentation\t\r \u00a0along\t\r \u00a0the\t\r \u00a0peptide\t\r \u00a0backbone\t\r \u00a0while\t\r \u00a0maintaining\t\r \u00a0the\t\r \u00a0phosphate\t\r \u00a0intact(426,\t\r \u00a0427).\t\r \u00a0\t\r \u00a0 1.3.6\t\r \u00a0Phosphopeptide\t\r \u00a0and\t\r \u00a0phosphoprotein\t\r \u00a0databases\t\r \u00a0As\t\r \u00a0the\t\r \u00a0last\t\r \u00a0five\t\r \u00a0years\t\r \u00a0have\t\r \u00a0seen\t\r \u00a0an\t\r \u00a0exponential\t\r \u00a0increase\t\r \u00a0in\t\r \u00a0the\t\r \u00a0number\t\r \u00a0of\t\r \u00a0identified\t\r \u00a0phosphorylation\t\r \u00a0sites,\t\r \u00a0several\t\r \u00a0groups\t\r \u00a0have\t\r \u00a0expended\t\r \u00a0enormous\t\r \u00a0efforts\t\r \u00a0to\t\r \u00a0curate\t\r \u00a0and\t\r \u00a0compile\t\r \u00a0these\t\r \u00a0data\t\r \u00a0into\t\r \u00a0on-\u00ad\u2010line\t\r \u00a0resources.\t\r \u00a0Much\t\r \u00a0of\t\r \u00a0the\t\r \u00a0high\t\r \u00a0quality\t\r \u00a0data\t\r \u00a0is\t\r \u00a0curated\t\r \u00a0in\t\r \u00a0UniProt(428).\t\r \u00a0In\t\r \u00a0addition,\t\r \u00a0more\t\r \u00a0specific\t\r \u00a0compendiums\t\r \u00a0are\t\r \u00a0available\t\r \u00a0and\t\r \u00a0each\t\r \u00a0provides\t\r \u00a0specific\t\r \u00a0types\t\r \u00a0of\t\r \u00a0tools\t\r \u00a0(e.g.,\t\r \u00a0predictors,\t\r \u00a0network\/pathway\t\r \u00a0viewers)\t\r \u00a0and\/or\t\r \u00a0information\t\r \u00a0(e.g.,\t\r \u00a0analytical\t\r \u00a0context\t\r \u00a0in\t\r \u00a0which\t\r \u00a0peptides\t\r \u00a0were\t\r \u00a0identified,\t\r \u00a0quantitative\t\r \u00a0profiles\t\r \u00a0of\t\r \u00a0phosphorylation\t\r \u00a0dynamics\t\r \u00a0after\t\r \u00a0agonist\t\r \u00a0stimulation).\t\r \u00a0Among\t\r \u00a0the\t\r \u00a0most\t\r \u00a0widely\t\r \u00a0used\t\r \u00a0and\t\r \u00a0largest\t\r \u00a0of\t\r \u00a0these\t\r \u00a0are\t\r \u00a0PhosphoNET\t\r \u00a0(www.phosphonet.ca),\t\r \u00a0Phosida\t\r \u00a0(www.phosida.com)\t\r \u00a0and\t\r \u00a0PhosphoSitePlus\t\r \u00a0(www.phosphosite.org),\t\r \u00a0each\t\r \u00a0of\t\r \u00a0which\t\r \u00a0currently\t\r \u00a0contains\t\r \u00a0over\t\r \u00a070,000\t\r \u00a0phosphosites.\t\r \u00a0Notably,\t\r \u00a0PhosphoNET\t\r \u00a0provides\t\r \u00a0information\t\r \u00a0regarding\t\r \u00a0experimentally\t\r \u00a0determined\t\r \u00a0kinases\t\r \u00a0and\t\r \u00a0phosphatases,\t\r \u00a0predicted\t\r \u00a0kinases\t\r \u00a0and\t\r \u00a0evolutionary\t\r \u00a0conservation,\t\r \u00a0and\t\r \u00a0Phosida\t\r \u00a0contains\t\r \u00a0MS\t\r \u00a0data\t\r \u00a0showing\t\r \u00a0temporal\t\r \u00a0regulation\t\r \u00a0of\t\r \u00a0phosphosites\t\r \u00a0by\t\r \u00a0various\t\r \u00a0stimuli(429).\t\r \u00a0\t\r \u00a0 1.4\t\r \u00a0Salmonella\t\r \u00a0and\t\r \u00a0proteomic\t\r \u00a0research\t\r \u00a0Several\t\r \u00a0studies\t\r \u00a0have\t\r \u00a0used\t\r \u00a02DE\t\r \u00a0to\t\r \u00a0compare\t\r \u00a0the\t\r \u00a0proteomes\t\r \u00a0of\t\r \u00a0various\t\r \u00a0 Salmonella\t\r \u00a0strains,\t\r \u00a0and\t\r \u00a0also\t\r \u00a0the\t\r \u00a0effects\t\r \u00a0of\t\r \u00a0numerous\t\r \u00a0environmental\t\r \u00a0conditions(430-\u00ad\u2010439).\t\r \u00a0A\t\r \u00a0fewer\t\r \u00a0number\t\r \u00a0of\t\r \u00a0similar\t\r \u00a0analyses\t\r \u00a0have\t\r \u00a0employed\t\r \u00a0techniques\t\r \u00a0in\t\r \u00a0LC-\u00ad\u2010MSn(440-\u00ad\u2010 \t\r \u00a0\t\r \u00a0 54\t\r \u00a0 443).\t\r \u00a0Unfortunately,\t\r \u00a0the\t\r \u00a0majority\t\r \u00a0of\t\r \u00a0these\t\r \u00a0have\t\r \u00a0utilized\t\r \u00a0in\t\r \u00a0vitro\t\r \u00a0culture\t\r \u00a0conditions,\t\r \u00a0while\t\r \u00a0only\t\r \u00a0a\t\r \u00a0couple\t\r \u00a0have\t\r \u00a0isolated\t\r \u00a0Salmonella\t\r \u00a0from\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0or\t\r \u00a0tissues.\t\r \u00a0These\t\r \u00a0include\t\r \u00a0purification\t\r \u00a0of\t\r \u00a0GFP-\u00ad\u2010tagged\t\r \u00a0S.\t\r \u00a0Typhimurium\t\r \u00a0from\t\r \u00a0infected\t\r \u00a0mouse\t\r \u00a0tissues\t\r \u00a0by\t\r \u00a0flow\t\r \u00a0cytometry,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0from\t\r \u00a0Raw\t\r \u00a0264.7\t\r \u00a0cells\t\r \u00a0at\t\r \u00a0various\t\r \u00a0times\t\r \u00a0post\t\r \u00a0infection(444,\t\r \u00a0445).\t\r \u00a0Interestingly,\t\r \u00a0the\t\r \u00a0latter\t\r \u00a0identified\t\r \u00a0a\t\r \u00a0novel\t\r \u00a0Salmonella\t\r \u00a0protein\t\r \u00a0required\t\r \u00a0for\t\r \u00a0replication\t\r \u00a0in\t\r \u00a0macrophages.\t\r \u00a0Furthermore,\t\r \u00a0only\t\r \u00a0a\t\r \u00a0few\t\r \u00a0analyses\t\r \u00a0have\t\r \u00a0focused\t\r \u00a0on\t\r \u00a0host\t\r \u00a0proteomes\t\r \u00a0during\t\r \u00a0infection,\t\r \u00a0while\t\r \u00a0the\t\r \u00a0majority\t\r \u00a0has\t\r \u00a0been\t\r \u00a0overwhelmingly\t\r \u00a0biased\t\r \u00a0towards\t\r \u00a0Salmonella\t\r \u00a0proteins.\t\r \u00a0A\t\r \u00a0study\t\r \u00a0identifying\t\r \u00a0proteins\t\r \u00a0from\t\r \u00a0Raw\t\r \u00a0264.7\t\r \u00a0cells\t\r \u00a0at\t\r \u00a0various\t\r \u00a0times\t\r \u00a0post\t\r \u00a0infection\t\r \u00a0by\t\r \u00a0S.\t\r \u00a0Typhimurium\t\r \u00a0observed\t\r \u00a0an\t\r \u00a0increase\t\r \u00a0in\t\r \u00a0mitochondrial\t\r \u00a0superoxide\t\r \u00a0dismutase\t\r \u00a0and\t\r \u00a0a\t\r \u00a0decrease\t\r \u00a0in\t\r \u00a0SNX6\t\r \u00a0which\t\r \u00a0was\t\r \u00a0SopB\t\r \u00a0dependent(446).\t\r \u00a0Protein\t\r \u00a0arrays\t\r \u00a0and\t\r \u00a0phosphospecific\t\r \u00a0antibodies\t\r \u00a0have\t\r \u00a0also\t\r \u00a0been\t\r \u00a0used\t\r \u00a0to\t\r \u00a0identify\t\r \u00a0host\t\r \u00a0proteins\t\r \u00a0phosphorylated\t\r \u00a0during\t\r \u00a0infection\t\r \u00a0by\t\r \u00a0S.\t\r \u00a0Typhimurium\t\r \u00a0strains\t\r \u00a0mutant\t\r \u00a0for\t\r \u00a0SopE\t\r \u00a0and\t\r \u00a0SopB(447).\t\r \u00a0Ultimately,\t\r \u00a0despite\t\r \u00a0the\t\r \u00a0relative\t\r \u00a0success\t\r \u00a0of\t\r \u00a0the\t\r \u00a0analyses\t\r \u00a0mentioned\t\r \u00a0above,\t\r \u00a0in\t\r \u00a0the\t\r \u00a0majority\t\r \u00a0of\t\r \u00a0cases\t\r \u00a0a\t\r \u00a0relatively\t\r \u00a0small\t\r \u00a0number\t\r \u00a0of\t\r \u00a0proteins\t\r \u00a0were\t\r \u00a0identified,\t\r \u00a0and\t\r \u00a0only\t\r \u00a0a\t\r \u00a0few\t\r \u00a0have\t\r \u00a0utilized\t\r \u00a0pre-\u00ad\u2010fractionation\t\r \u00a0techniques,\t\r \u00a0while\t\r \u00a0none\t\r \u00a0have\t\r \u00a0employed\t\r \u00a0quantitative\t\r \u00a0or\t\r \u00a0PTM\t\r \u00a0analyses\t\r \u00a0by\t\r \u00a0LC-\u00ad\u2010MSn(448-\u00ad\u2010450).\t\r \u00a0\t\r \u00a0 1.5\t\r \u00a0Research\t\r \u00a0aims\t\r \u00a0and\t\r \u00a0hypothesis\t\r \u00a0The\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0effector\t\r \u00a0SopB\t\r \u00a0localizes\t\r \u00a0to\t\r \u00a0both\t\r \u00a0the\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0membrane\t\r \u00a0and\t\r \u00a0the\t\r \u00a0SCV,\t\r \u00a0and\t\r \u00a0has\t\r \u00a0been\t\r \u00a0reported\t\r \u00a0to\t\r \u00a0have\t\r \u00a0a\t\r \u00a0vast\t\r \u00a0and\t\r \u00a0broad\t\r \u00a0impact\t\r \u00a0on\t\r \u00a0several\t\r \u00a0intracellular\t\r \u00a0aspects\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0infection.\t\r \u00a0These\t\r \u00a0include\t\r \u00a0the\t\r \u00a0induction\t\r \u00a0of\t\r \u00a0innate\t\r \u00a0immune\t\r \u00a0responses\t\r \u00a0in\t\r \u00a0epithelial\t\r \u00a0cells\t\r \u00a0and\t\r \u00a0biogenesis\t\r \u00a0and\t\r \u00a0maturation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV,\t\r \u00a0both\t\r \u00a0of\t\r \u00a0which\t\r \u00a0are\t\r \u00a0central\t\r \u00a0to\t\r \u00a0Salmonella\t\r \u00a0virulence.\t\r \u00a0The\t\r \u00a0hypothesis\t\r \u00a0of\t\r \u00a0this\t\r \u00a0thesis\t\r \u00a0is\t\r \u00a0that\t\r \u00a0several\t\r \u00a0 \t\r \u00a0\t\r \u00a0 55\t\r \u00a0 currently\t\r \u00a0unidentified\t\r \u00a0host\t\r \u00a0proteins\t\r \u00a0are\t\r \u00a0targeted\t\r \u00a0both\t\r \u00a0directly\t\r \u00a0and\t\r \u00a0indirectly\t\r \u00a0by\t\r \u00a0SopB\t\r \u00a0during\t\r \u00a0infection,\t\r \u00a0and\t\r \u00a0that\t\r \u00a0these\t\r \u00a0play\t\r \u00a0a\t\r \u00a0significant\t\r \u00a0role\t\r \u00a0in\t\r \u00a0Salmonella\t\r \u00a0pathogenesis\t\r \u00a0and\t\r \u00a0disease.\t\r \u00a0A\t\r \u00a0combination\t\r \u00a0of\t\r \u00a0mass-\u00ad\u2010spectrometry\t\r \u00a0based\t\r \u00a0proteomics\t\r \u00a0and\t\r \u00a0bioinformatics\t\r \u00a0techniques\t\r \u00a0have\t\r \u00a0been\t\r \u00a0employed\t\r \u00a0with\t\r \u00a0the\t\r \u00a0aim\t\r \u00a0of\t\r \u00a0identifying\t\r \u00a0these\t\r \u00a0host\t\r \u00a0targets.\t\r \u00a0These\t\r \u00a0include\t\r \u00a0SILAC\t\r \u00a0for\t\r \u00a0quantitation,\t\r \u00a0immunoprecipitation,\t\r \u00a0SDS-\u00ad\u2010PAGE\t\r \u00a0and\t\r \u00a0IEF\t\r \u00a0for\t\r \u00a0pre-\u00ad\u2010fractionation,\t\r \u00a0and\t\r \u00a0several\t\r \u00a0techniques\t\r \u00a0that\t\r \u00a0have\t\r \u00a0been\t\r \u00a0developed\t\r \u00a0here\t\r \u00a0for\t\r \u00a0the\t\r \u00a0global\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0protein\t\r \u00a0phosphorylation\t\r \u00a0events\t\r \u00a0induced\t\r \u00a0in\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0during\t\r \u00a0Salmonella\t\r \u00a0infection.\t\r \u00a0 \t\r \u00a0\t\r \u00a0 56\t\r \u00a0 2\t\r \u00a0Identification\t\r \u00a0of\t\r \u00a0cognate\t\r \u00a0host\t\r \u00a0targets\t\r \u00a0and\t\r \u00a0specific\t\r \u00a0 ubiquitylation\t\r \u00a0sites\t\r \u00a0on\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0effector\t\r \u00a0 SopB\/SigD\t\r \u00a0 2.1\t\r \u00a0Introduction\t\r \u00a0 Salmonella\t\r \u00a0is\t\r \u00a0a\t\r \u00a0food-\u00ad\u2010borne,\t\r \u00a0facultative\t\r \u00a0intracellular\t\r \u00a0bacterium\t\r \u00a0that\t\r \u00a0causes\t\r \u00a0gastroenteritis(451)\t\r \u00a0and\t\r \u00a0typhoid\t\r \u00a0fever\t\r \u00a0in\t\r \u00a0humans(452).\t\r \u00a0The\t\r \u00a0various\t\r \u00a0serovars\t\r \u00a0of\t\r \u00a0S.\t\r \u00a0 enterica\t\r \u00a0have\t\r \u00a0evolved\t\r \u00a0a\t\r \u00a0sophisticated\t\r \u00a0mechanism\t\r \u00a0for\t\r \u00a0evading\t\r \u00a0host\t\r \u00a0immunity\t\r \u00a0whereby\t\r \u00a0the\t\r \u00a0pathogen\t\r \u00a0induces\t\r \u00a0its\t\r \u00a0own\t\r \u00a0internalization\t\r \u00a0into\t\r \u00a0a\t\r \u00a0membrane-\u00ad\u2010bound\t\r \u00a0vacuole\t\r \u00a0termed\t\r \u00a0the\t\r \u00a0SCV(451-\u00ad\u2010453).\t\r \u00a0Formation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV\t\r \u00a0is\t\r \u00a0determined\t\r \u00a0by\t\r \u00a0effector\t\r \u00a0proteins,\t\r \u00a0which\t\r \u00a0are\t\r \u00a0translocated\t\r \u00a0into\t\r \u00a0the\t\r \u00a0host\t\r \u00a0cytosol\t\r \u00a0by\t\r \u00a0two\t\r \u00a0T3SS\t\r \u00a0encoded\t\r \u00a0on\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0and\t\r \u00a0SPI-\u00ad\u20102(453-\u00ad\u2010455).\t\r \u00a0Translocated\t\r \u00a0upon\t\r \u00a0contact\t\r \u00a0with\t\r \u00a0host\t\r \u00a0cells,\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0effectors\t\r \u00a0modulate\t\r \u00a0Rho\t\r \u00a0family\t\r \u00a0GTPases\t\r \u00a0to\t\r \u00a0mediate\t\r \u00a0specific\t\r \u00a0activities\t\r \u00a0such\t\r \u00a0as\t\r \u00a0actin\t\r \u00a0remodeling,\t\r \u00a0macropinocytosis,\t\r \u00a0and\t\r \u00a0nuclear\t\r \u00a0responses\t\r \u00a0within\t\r \u00a0the\t\r \u00a0host\t\r \u00a0cell(45,\t\r \u00a0456,\t\r \u00a0457).\t\r \u00a0More\t\r \u00a0specifically,\t\r \u00a0effectors\t\r \u00a0SopE\t\r \u00a0and\t\r \u00a0SopE2\t\r \u00a0are\t\r \u00a0GEFs\t\r \u00a0for\t\r \u00a0the\t\r \u00a0Rho\t\r \u00a0family\t\r \u00a0GTPases\t\r \u00a0Rac1\t\r \u00a0and\t\r \u00a0Cdc42,\t\r \u00a0which\t\r \u00a0cause\t\r \u00a0actin\t\r \u00a0polymerization\t\r \u00a0and\t\r \u00a0membrane\t\r \u00a0ruffling\t\r \u00a0(Rac1),\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0nuclear\t\r \u00a0responses\t\r \u00a0and\t\r \u00a0cytokine\t\r \u00a0production\t\r \u00a0(Cdc42)(45,\t\r \u00a0187).\t\r \u00a0Similarly,\t\r \u00a0the\t\r \u00a0effector\t\r \u00a0SopB\t\r \u00a0(also\t\r \u00a0called\t\r \u00a0SigD)\t\r \u00a0induces\t\r \u00a0membrane\t\r \u00a0ruffling\t\r \u00a0and\t\r \u00a0macropinocytosis\t\r \u00a0through\t\r \u00a0the\t\r \u00a0indirect\t\r \u00a0activation\t\r \u00a0of\t\r \u00a0SGEF,\t\r \u00a0and\t\r \u00a0also\t\r \u00a0activates\t\r \u00a0Cdc42\t\r \u00a0through\t\r \u00a0an\t\r \u00a0undefined\t\r \u00a0mechanism(45,\t\r \u00a0187).\t\r \u00a0Finally,\t\r \u00a0the\t\r \u00a0effector\t\r \u00a0SptP\t\r \u00a0acts\t\r \u00a0as\t\r \u00a0a\t\r \u00a0GAP,\t\r \u00a0inactivating\t\r \u00a0Rac1\t\r \u00a0and\t\r \u00a0Cdc42(457).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 57\t\r \u00a0 In\t\r \u00a0addition\t\r \u00a0to\t\r \u00a0its\t\r \u00a0effects\t\r \u00a0on\t\r \u00a0macropinocytosis\t\r \u00a0and\t\r \u00a0nuclear\t\r \u00a0activity,\t\r \u00a0several\t\r \u00a0other\t\r \u00a0structural\t\r \u00a0and\t\r \u00a0functional\t\r \u00a0features\t\r \u00a0of\t\r \u00a0SopB\t\r \u00a0have\t\r \u00a0been\t\r \u00a0identified.\t\r \u00a0It\t\r \u00a0has\t\r \u00a0a\t\r \u00a0membrane-\u00ad\u2010targeting\t\r \u00a0domain\t\r \u00a0near\t\r \u00a0the\t\r \u00a0amino\t\r \u00a0terminus,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0a\t\r \u00a0region\t\r \u00a0that\t\r \u00a0negatively\t\r \u00a0affects\t\r \u00a0actin\t\r \u00a0polymerization(458,\t\r \u00a0459).\t\r \u00a0We,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0others,\t\r \u00a0have\t\r \u00a0previously\t\r \u00a0shown\t\r \u00a0that\t\r \u00a0SopB\t\r \u00a0contains\t\r \u00a0multiple\t\r \u00a0regions\t\r \u00a0with\t\r \u00a0homology\t\r \u00a0to\t\r \u00a0phosphoinositide\t\r \u00a0phosphatases,\t\r \u00a0and\t\r \u00a0that\t\r \u00a0it\t\r \u00a0is\t\r \u00a0capable\t\r \u00a0of\t\r \u00a0dephosphorylating\t\r \u00a0phosphoinositides\t\r \u00a0in\t\r \u00a0the\t\r \u00a04\t\r \u00a0and\t\r \u00a05\t\r \u00a0positions(163,\t\r \u00a0185).\t\r \u00a0Through\t\r \u00a0their\t\r \u00a0catalytic\t\r \u00a0activity,\t\r \u00a0these\t\r \u00a0regions\t\r \u00a0have\t\r \u00a0been\t\r \u00a0implicated\t\r \u00a0in\t\r \u00a0processes\t\r \u00a0ranging\t\r \u00a0from\t\r \u00a0SCV\t\r \u00a0trafficking(57,\t\r \u00a0200),\t\r \u00a0membrane\t\r \u00a0fission(49,\t\r \u00a0460),\t\r \u00a0Akt\t\r \u00a0activation(163,\t\r \u00a0461),\t\r \u00a0and\t\r \u00a0increased\t\r \u00a0chloride\t\r \u00a0ion\t\r \u00a0secretion\t\r \u00a0from\t\r \u00a0epithelial\t\r \u00a0cells(164).\t\r \u00a0While\t\r \u00a0these\t\r \u00a0activities\t\r \u00a0are\t\r \u00a0dependent\t\r \u00a0on\t\r \u00a0catalytic\t\r \u00a0activity\t\r \u00a0and\t\r \u00a0an\t\r \u00a0indirect\t\r \u00a0link\t\r \u00a0has\t\r \u00a0been\t\r \u00a0made\t\r \u00a0between\t\r \u00a0SopB\t\r \u00a0and\t\r \u00a0RhoG\t\r \u00a0activation,\t\r \u00a0the\t\r \u00a0means\t\r \u00a0by\t\r \u00a0which\t\r \u00a0SopB\t\r \u00a0activates\t\r \u00a0Cdc42,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0other\t\r \u00a0possible\t\r \u00a0substrates\t\r \u00a0of\t\r \u00a0this\t\r \u00a0phosphatase,\t\r \u00a0remain\t\r \u00a0largely\t\r \u00a0unknown.\t\r \u00a0In\t\r \u00a0recent\t\r \u00a0work,\t\r \u00a0Rodr\u00edguez-\u00ad\u2010Escudero\t\r \u00a0et\t\r \u00a0al.\t\r \u00a0have\t\r \u00a0shown\t\r \u00a0Cdc42\t\r \u00a0from\t\r \u00a0a\t\r \u00a0Saccharomyces\t\r \u00a0cerevisiae\t\r \u00a0lysate\t\r \u00a0to\t\r \u00a0co-\u00ad\u2010purify\t\r \u00a0with\t\r \u00a0catalytically\t\r \u00a0inactive\t\r \u00a0GST-\u00ad\u2010SopB\t\r \u00a0but\t\r \u00a0not\t\r \u00a0a\t\r \u00a0GST-\u00ad\u2010SopB\t\r \u00a0protein\t\r \u00a0lacking\t\r \u00a0a\t\r \u00a0region\t\r \u00a0previously\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0affect\t\r \u00a0actin\t\r \u00a0polymerization\t\r \u00a0and\t\r \u00a0Cdc42\t\r \u00a0signaling(462).\t\r \u00a0However,\t\r \u00a0while\t\r \u00a0this\t\r \u00a0study\t\r \u00a0has\t\r \u00a0defined\t\r \u00a0a\t\r \u00a0SopB\t\r \u00a0region\t\r \u00a0responsible\t\r \u00a0for\t\r \u00a0co-\u00ad\u2010purification\t\r \u00a0of\t\r \u00a0Cdc42\t\r \u00a0in\t\r \u00a0yeast,\t\r \u00a0further\t\r \u00a0work\t\r \u00a0is\t\r \u00a0required\t\r \u00a0to\t\r \u00a0determine\t\r \u00a0if\t\r \u00a0the\t\r \u00a0interaction\t\r \u00a0is\t\r \u00a0direct,\t\r \u00a0and\t\r \u00a0whether\t\r \u00a0it\t\r \u00a0occurs\t\r \u00a0in\t\r \u00a0a\t\r \u00a0more\t\r \u00a0physiological\t\r \u00a0setting.\t\r \u00a0Technical\t\r \u00a0and\t\r \u00a0analytical\t\r \u00a0developments\t\r \u00a0in\t\r \u00a0the\t\r \u00a0last\t\r \u00a0few\t\r \u00a0years\t\r \u00a0have\t\r \u00a0allowed\t\r \u00a0quantitative\t\r \u00a0proteomics\t\r \u00a0to\t\r \u00a0become\t\r \u00a0a\t\r \u00a0powerful\t\r \u00a0tool\t\r \u00a0for\t\r \u00a0elucidating\t\r \u00a0dynamic\t\r \u00a0changes\t\r \u00a0in\t\r \u00a0organelles\t\r \u00a0and\t\r \u00a0protein-\u00ad\u2010protein\t\r \u00a0interactions(333,\t\r \u00a0463).\t\r \u00a0One\t\r \u00a0particular\t\r \u00a0method,\t\r \u00a0termed\t\r \u00a0SILAC\t\r \u00a0(330)\t\r \u00a0is\t\r \u00a0now\t\r \u00a0a\t\r \u00a0widespread\t\r \u00a0tool\t\r \u00a0for\t\r \u00a0metabolically\t\r \u00a0incorporating\t\r \u00a0 \t\r \u00a0\t\r \u00a0 58\t\r \u00a0 isotopic\t\r \u00a0labels\t\r \u00a0into\t\r \u00a0protein\t\r \u00a0samples\t\r \u00a0for\t\r \u00a0relative\t\r \u00a0quantification.\t\r \u00a0SILAC\t\r \u00a0is\t\r \u00a0particularly\t\r \u00a0useful\t\r \u00a0in\t\r \u00a0differentiating\t\r \u00a0specific\t\r \u00a0from\t\r \u00a0non-\u00ad\u2010specific\t\r \u00a0binding\t\r \u00a0partners\t\r \u00a0during\t\r \u00a0protein\t\r \u00a0interaction\t\r \u00a0studies.\t\r \u00a0This\t\r \u00a0is\t\r \u00a0achieved\t\r \u00a0by\t\r \u00a0differentially\t\r \u00a0labeling\t\r \u00a0two\t\r \u00a0cell\t\r \u00a0cultures\t\r \u00a0and\t\r \u00a0affinity\t\r \u00a0purifying\t\r \u00a0the\t\r \u00a0bait\t\r \u00a0from\t\r \u00a0one\t\r \u00a0population\t\r \u00a0and\t\r \u00a0a\t\r \u00a0control\t\r \u00a0from\t\r \u00a0the\t\r \u00a0other.\t\r \u00a0In\t\r \u00a0this\t\r \u00a0case,\t\r \u00a0when\t\r \u00a0the\t\r \u00a0isolated\t\r \u00a0complexes\t\r \u00a0are\t\r \u00a0mixed\t\r \u00a0and\t\r \u00a0analyzed,\t\r \u00a0background\t\r \u00a0proteins\t\r \u00a0bind\t\r \u00a0equally\t\r \u00a0to\t\r \u00a0the\t\r \u00a0bait\t\r \u00a0and\t\r \u00a0control\t\r \u00a0resulting\t\r \u00a0in\t\r \u00a0one-\u00ad\u2010to-\u00ad\u2010one\t\r \u00a0light-\u00ad\u2010to-\u00ad\u2010heavy\t\r \u00a0ratios,\t\r \u00a0while\t\r \u00a0specific\t\r \u00a0interactions\t\r \u00a0with\t\r \u00a0the\t\r \u00a0bait\t\r \u00a0result\t\r \u00a0in\t\r \u00a0differential\t\r \u00a0ratios(464,\t\r \u00a0465).\t\r \u00a0In\t\r \u00a0the\t\r \u00a0current\t\r \u00a0study,\t\r \u00a0a\t\r \u00a0SILAC\t\r \u00a0screen\t\r 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\u00a0expression\t\r \u00a0plasmid\t\r \u00a0expressing\t\r \u00a0an\t\r \u00a0N-\u00ad\u2010terminal\t\r \u00a0GST\t\r \u00a0tagged\t\r \u00a0fusion\t\r \u00a0of\t\r \u00a0SopB\t\r \u00a0was\t\r \u00a0constructed\t\r \u00a0as\t\r \u00a0follows:\t\r \u00a0the\t\r \u00a0entire\t\r \u00a0SopB\t\r \u00a0coding\t\r \u00a0region\t\r \u00a0was\t\r \u00a0amplified\t\r \u00a0from\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0Typhimurium\t\r \u00a0SL1344\t\r \u00a0genomic\t\r \u00a0DNA\t\r \u00a0with\t\r \u00a0Phusion\t\r \u00a0High-\u00ad\u2010Fidelity\t\r \u00a0DNA\t\r \u00a0Polymerase\t\r \u00a0(New\t\r \u00a0England\t\r \u00a0Biolabs,\t\r \u00a0Pickering,\t\r \u00a0ON,\t\r \u00a0Canada)\t\r \u00a0using\t\r \u00a0the\t\r \u00a0primers\t\r \u00a0SopB5\t\r \u00a0(5\u2019-\u00ad\u2010GGA\t\r \u00a0TGC\t\r \u00a0AAA\t\r \u00a0TAC\t\r \u00a0AGA\t\r \u00a0GCT\t\r \u00a0TCT\t\r \u00a0ATC-\u00ad\u20103\u2019)\t\r \u00a0and\t\r \u00a0SopB3-\u00ad\u2010NotI\t\r 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\u00a0from\t\r \u00a0pSopBD28\t\r \u00a0and\t\r \u00a0pSopBD28(C462S)\t\r \u00a0into\t\r \u00a0the\t\r \u00a0corresponding\t\r \u00a0sites\t\r \u00a0of\t\r \u00a0pEGFP-\u00ad\u2010C3\t\r \u00a0(Clontech,\t\r \u00a0Mountainview,\t\r \u00a0CA).\t\r \u00a0\t\r \u00a0 2.2.2\t\r \u00a0Preparation\t\r \u00a0of\t\r \u00a0GST-\u00ad\u2010SopB\t\r \u00a0protein\t\r \u00a0The\t\r \u00a0pGEX-\u00ad\u2010SopB\t\r \u00a0construct\t\r \u00a0was\t\r \u00a0transformed\t\r \u00a0into\t\r \u00a0competent\t\r \u00a0BL21(DE3)pLysS\t\r \u00a0 E.\t\r \u00a0coli\t\r \u00a0cells\t\r \u00a0(Stratagene).\t\r \u00a0A\t\r \u00a0single\t\r \u00a0colony\t\r \u00a0was\t\r \u00a0picked\t\r \u00a0to\t\r \u00a0initiate\t\r \u00a0a\t\r \u00a05\t\r \u00a0mL\t\r \u00a0overnight\t\r \u00a0culture\t\r \u00a0grown\t\r \u00a0in\t\r \u00a02xYT\t\r \u00a0bacterial\t\r \u00a0growth\t\r \u00a0media\t\r \u00a0at\t\r \u00a037\u00b0C.\t\r \u00a0The\t\r \u00a0overnight\t\r \u00a0culture\t\r \u00a0was\t\r \u00a0then\t\r \u00a0used\t\r \u00a0to\t\r \u00a0inoculate\t\r \u00a0a\t\r \u00a0500\t\r \u00a0mL\t\r \u00a0culture\t\r \u00a0in\t\r \u00a02xYT\t\r \u00a0media\t\r \u00a0which\t\r \u00a0was\t\r \u00a0grown\t\r \u00a0at\t\r \u00a028\u00b0C\t\r \u00a0to\t\r \u00a0an\t\r \u00a0OD600\t\r \u00a0of\t\r \u00a0between\t\r \u00a00.6-\u00ad\u20100.8.\t\r \u00a0Expression\t\r \u00a0of\t\r \u00a0the\t\r \u00a0GST-\u00ad\u2010SopB\t\r \u00a0recombinant\t\r \u00a0protein\t\r \u00a0was\t\r \u00a0induced\t\r \u00a0by\t\r \u00a0addition\t\r \u00a0of\t\r \u00a00.05\t\r \u00a0mM\t\r \u00a0isopropyl\t\r \u00a0\u03b2-\u00ad\u2010D-\u00ad\u20101-\u00ad\u2010thiogalactopyranoside\t\r \u00a0and\t\r \u00a0the\t\r \u00a0culture\t\r \u00a0was\t\r \u00a0grown\t\r \u00a0for\t\r \u00a0an\t\r \u00a0additional\t\r \u00a02\t\r \u00a0to\t\r \u00a02.5\t\r \u00a0hrs\t\r \u00a0at\t\r \u00a028\u00b0C.\t\r \u00a0Cells\t\r \u00a0were\t\r \u00a0harvested\t\r \u00a0by\t\r \u00a0centrifugation,\t\r \u00a0re-\u00ad\u2010suspended\t\r \u00a0and\t\r \u00a0lysed\t\r \u00a0by\t\r \u00a0sonication\t\r \u00a0(3\t\r \u00a0x\t\r \u00a010\t\r \u00a0sec)\t\r \u00a0in\t\r \u00a0phosphate-\u00ad\u2010buffered\t\r \u00a0saline\t\r \u00a0(PBS)\t\r \u00a0containing\t\r \u00a02\t\r \u00a0mM\t\r \u00a0EDTA\t\r \u00a0and\t\r \u00a0protease\t\r \u00a0inhibitors\t\r \u00a0(Complete\t\r \u00a0 \t\r \u00a0\t\r \u00a0 61\t\r \u00a0 tablets,\t\r \u00a0Roche\t\r \u00a0Diagnostics),\t\r \u00a0then\t\r \u00a0equilibrated\t\r \u00a0to\t\r \u00a01%\t\r \u00a0Triton\t\r \u00a0X-\u00ad\u2010100\t\r \u00a0followed\t\r \u00a0by\t\r \u00a0incubation\t\r \u00a0at\t\r \u00a04\u00b0C\t\r \u00a0for\t\r \u00a01\t\r \u00a0to\t\r \u00a02\t\r \u00a0hr\t\r \u00a0in\t\r \u00a0order\t\r \u00a0to\t\r \u00a0increase\t\r \u00a0yield\t\r \u00a0of\t\r \u00a0soluble\t\r \u00a0recombinant\t\r \u00a0protein.\t\r \u00a0The\t\r \u00a0lysate\t\r \u00a0was\t\r \u00a0cleared\t\r \u00a0by\t\r \u00a0centrifugation\t\r \u00a0and\t\r \u00a0the\t\r \u00a0supernatant\t\r \u00a0incubated\t\r \u00a0at\t\r \u00a04\u00b0C\t\r \u00a0for\t\r \u00a01\t\r \u00a0to\t\r \u00a02\t\r \u00a0hrs\t\r \u00a0with\t\r \u00a0glutathione-\u00ad\u2010agarose\t\r \u00a0beads\t\r \u00a0(Sigma-\u00ad\u2010Aldrich).\t\r \u00a0Beads\t\r \u00a0and\t\r \u00a0associated\t\r \u00a0proteins\t\r \u00a0were\t\r \u00a0first\t\r \u00a0washed\t\r \u00a0with\t\r \u00a0Wash\t\r \u00a0Buffer\t\r \u00a01\t\r \u00a0(PBS,\t\r \u00a02\t\r \u00a0mM\t\r \u00a0EDTA,\t\r \u00a01%\t\r \u00a0Triton\t\r \u00a0X-\u00ad\u2010100)\t\r \u00a0and\t\r \u00a0then\t\r \u00a0subsequently\t\r \u00a0with\t\r \u00a0Wash\t\r \u00a0Buffer\t\r \u00a02\t\r \u00a0(50\t\r \u00a0mM\t\r \u00a0Tris-\u00ad\u2010Cl,\t\r \u00a0150\t\r \u00a0mM\t\r \u00a0NaCl,\t\r \u00a0pH\t\r \u00a08.0).\t\r \u00a0\t\r \u00a0GST-\u00ad\u2010SopB\t\r \u00a0protein\t\r \u00a0was\t\r \u00a0eluted\t\r \u00a0using\t\r \u00a0free,\t\r \u00a0reduced\t\r \u00a0glutathione\t\r \u00a0(10\t\r \u00a0mM\t\r \u00a0in\t\r \u00a0Wash\t\r \u00a0Buffer\t\r \u00a02)\t\r \u00a0and\t\r \u00a0then\t\r \u00a0dialyzed\t\r \u00a0against\t\r \u00a0Wash\t\r \u00a0Buffer\t\r \u00a02\t\r \u00a0containing\t\r \u00a0no\t\r \u00a0glutathione.\t\r \u00a0The\t\r \u00a0recombinant\t\r \u00a0protein\t\r \u00a0was\t\r \u00a0rebound\t\r \u00a0to\t\r \u00a0fresh\t\r \u00a0glutathione-\u00ad\u2010agarose\t\r \u00a0beads\t\r \u00a0and\t\r \u00a0the\t\r \u00a0amount\t\r \u00a0of\t\r \u00a0recombinant\t\r \u00a0protein\t\r \u00a0bound\t\r \u00a0was\t\r \u00a0estimated\t\r \u00a0by\t\r \u00a0visual\t\r \u00a0inspection\t\r \u00a0of\t\r \u00a0an\t\r \u00a0SDS-\u00ad\u2010PAGE\t\r \u00a0gel.\t\r \u00a0The\t\r \u00a0control\t\r \u00a0GST\t\r \u00a0protein\t\r \u00a0was\t\r \u00a0prepared\t\r \u00a0and\t\r \u00a0concentration\t\r \u00a0estimated\t\r \u00a0in\t\r \u00a0parallel.\t\r \u00a0\t\r \u00a0 2.2.3\t\r \u00a0Cell\t\r \u00a0culture\t\r \u00a0and\t\r \u00a0transfections\t\r \u00a0HEK293\t\r \u00a0and\t\r \u00a0HeLa\t\r \u00a0cells\t\r \u00a0were\t\r \u00a0maintained\t\r \u00a0in\t\r \u00a0Dulbecco\u2019s\t\r \u00a0Modified\t\r \u00a0Eagle\t\r \u00a0Medium\t\r \u00a0(DMEM)\t\r \u00a0containing\t\r \u00a04500mg\/L\t\r \u00a0glucose\t\r \u00a0and\t\r \u00a04mM\t\r \u00a0L-\u00ad\u2010glutamine\t\r \u00a0(Thermo\t\r \u00a0Fisher\t\r \u00a0Scientific,\t\r \u00a0Ottawa,\t\r \u00a0ON,\t\r \u00a0Canada),\t\r \u00a0supplemented\t\r \u00a0with\t\r \u00a010%\t\r \u00a0qualified\t\r \u00a0fetal\t\r \u00a0bovine\t\r \u00a0serum\t\r \u00a0(FBS)(Invitrogen),\t\r \u00a0an\t\r \u00a0additional\t\r \u00a02\t\r \u00a0mM\t\r \u00a0L-\u00ad\u2010glutamine\t\r \u00a0(Thermo\t\r \u00a0Fisher\t\r \u00a0Scientific)\t\r \u00a0and\t\r \u00a00.1\t\r \u00a0units\/L\t\r \u00a0penicillin\t\r \u00a0and\t\r \u00a0streptomycin\t\r \u00a0(Thermo\t\r \u00a0Fisher\t\r \u00a0Scientific).\t\r \u00a0Raw264.7\t\r \u00a0cells\t\r \u00a0were\t\r \u00a0maintained\t\r \u00a0as\t\r \u00a0described\t\r \u00a0above\t\r \u00a0except\t\r \u00a0the\t\r \u00a0FBS\t\r \u00a0was\t\r \u00a0heat-\u00ad\u2010treated\t\r \u00a0at\t\r \u00a056\u02daC\t\r \u00a0for\t\r \u00a045\t\r \u00a0min\t\r \u00a0prior\t\r \u00a0to\t\r \u00a0use.\t\r \u00a0For\t\r \u00a0SILAC\t\r \u00a0labeling,\t\r \u00a0cells\t\r \u00a0were\t\r \u00a0split\t\r \u00a0from\t\r \u00a0normal\t\r \u00a0growth\t\r \u00a0media\t\r \u00a0into\t\r \u00a0arginine\t\r \u00a0and\t\r \u00a0lysine-\u00ad\u2010free\t\r \u00a0DMEM\t\r \u00a0(Caisson\t\r \u00a0Laboratories\t\r \u00a0 \t\r \u00a0\t\r \u00a0 62\t\r \u00a0 Inc.)\t\r \u00a0supplemented\t\r \u00a0with\t\r \u00a010%\t\r \u00a0dialyzed\t\r \u00a0FBS\t\r \u00a0(Invitrogen),\t\r \u00a00.1\t\r \u00a0units\/L\t\r \u00a0penicillin\t\r \u00a0and\t\r \u00a0streptomycin\t\r \u00a0and\t\r \u00a0either\t\r \u00a036.5\t\r \u00a0mg\/L\t\r \u00a0D4-\u00ad\u2010lysine\t\r \u00a0and\t\r \u00a021\t\r \u00a0mg\/L\t\r \u00a013C6-\u00ad\u2010arginine\t\r \u00a0(Cambridge\t\r \u00a0Isotope\t\r \u00a0Laboratories,\t\r \u00a0Andover,\t\r \u00a0MA)\t\r \u00a0for\t\r \u00a0heavy\t\r \u00a0cells\t\r \u00a0or\t\r \u00a0equimolar\t\r \u00a0amounts\t\r \u00a0of\t\r \u00a0normal\t\r \u00a0isotopic\t\r \u00a0abundance\t\r \u00a0L-\u00ad\u2010arginine\t\r \u00a0and\t\r \u00a0L-\u00ad\u2010lysine\t\r \u00a0(Sigma-\u00ad\u2010Aldrich,\t\r \u00a0Oakville,\t\r \u00a0ON)\t\r \u00a0for\t\r \u00a0light\t\r \u00a0cells.\t\r \u00a0The\t\r \u00a0cells\t\r \u00a0were\t\r \u00a0then\t\r \u00a0maintained\t\r \u00a0in\t\r \u00a0labeling\t\r \u00a0media\t\r \u00a0for\t\r \u00a0at\t\r \u00a0least\t\r \u00a06\t\r \u00a0cell\t\r \u00a0divisions,\t\r \u00a0as\t\r \u00a0described(330).\t\r \u00a0In\t\r \u00a0initial\t\r \u00a0testing\t\r \u00a0we\t\r \u00a0found\t\r \u00a0that\t\r \u00a0growing\t\r \u00a0HEK293\t\r \u00a0cells\t\r \u00a0in\t\r \u00a0the\t\r \u00a0dialyzed\t\r \u00a0FBS\t\r \u00a0required\t\r \u00a0for\t\r \u00a0SILAC\t\r \u00a0labeling\t\r \u00a0eliminated\t\r \u00a0their\t\r \u00a0transfectability.\t\r \u00a0In\t\r \u00a0normal\t\r \u00a0FBS\t\r \u00a0approximately\t\r \u00a050%\t\r \u00a0of\t\r \u00a0cells\t\r \u00a0took\t\r \u00a0up\t\r \u00a0a\t\r \u00a0control\t\r \u00a0vector\t\r \u00a0encoding\t\r \u00a0GFP\t\r \u00a0but\t\r \u00a0we\t\r \u00a0were\t\r \u00a0unable\t\r \u00a0to\t\r \u00a0find\t\r \u00a0any\t\r \u00a0evidence\t\r \u00a0of\t\r \u00a0transfection\t\r \u00a0in\t\r \u00a0cells\t\r \u00a0grown\t\r \u00a0in\t\r \u00a0dialyzed\t\r \u00a0FBS.\t\r \u00a0Therefore,\t\r \u00a0for\t\r \u00a0SILAC\t\r \u00a0transfection\t\r \u00a0experiments,\t\r \u00a0cells\t\r \u00a0grown\t\r \u00a0in\t\r \u00a0normal\t\r \u00a0media\t\r \u00a0were\t\r \u00a0transfected\t\r \u00a0and\t\r \u00a0compared\t\r \u00a0to\t\r \u00a0non-\u00ad\u2010specific\t\r \u00a0binding\t\r \u00a0from\t\r \u00a0heavy\t\r \u00a0SILAC\t\r \u00a0cells.\t\r \u00a0For\t\r \u00a0all\t\r \u00a0transfections\t\r \u00a0cells\t\r \u00a0were\t\r \u00a0grown\t\r \u00a0to\t\r \u00a0approximately\t\r \u00a030%\t\r \u00a0confluency\t\r \u00a0on\t\r \u00a010\t\r \u00a0cm\t\r \u00a0diameter\t\r \u00a0cell\t\r \u00a0culture\t\r \u00a0plates\t\r \u00a0or\t\r \u00a06-\u00ad\u2010well\t\r \u00a0plates\t\r \u00a0containing\t\r \u00a012\t\r \u00a0mm\t\r \u00a0diameter\t\r \u00a0glass\t\r \u00a0cover\t\r \u00a0slips.\t\r \u00a0Cells\t\r \u00a0were\t\r \u00a0transfected\t\r \u00a0for\t\r \u00a018-\u00ad\u201024\t\r \u00a0h\t\r \u00a0with\t\r \u00a08\t\r \u00a0\u03bcg\t\r \u00a0of\t\r \u00a0each\t\r \u00a0plasmid\t\r \u00a0per\t\r \u00a010\t\r \u00a0cm\t\r \u00a0plate,\t\r \u00a0and\t\r \u00a01.3\t\r \u00a0\u03bcg\t\r \u00a0per\t\r \u00a0well\t\r \u00a0using\t\r \u00a0calcium\t\r \u00a0phosphate\t\r \u00a0as\t\r \u00a0described(466).\t\r \u00a0\t\r \u00a0 2.2.4\t\r \u00a0SILAC\t\r \u00a02HA-\u00ad\u2010SopB\t\r \u00a0immunoprecipitations\t\r \u00a0Ten\t\r \u00a010\t\r \u00a0cm\t\r \u00a0cell\t\r \u00a0culture\t\r \u00a0plates\t\r \u00a0of\t\r \u00a0unlabeled\t\r \u00a0HEK293\t\r \u00a0cells\t\r \u00a0were\t\r \u00a0transfected\t\r \u00a0with\t\r \u00a02HA-\u00ad\u2010SopB\t\r \u00a0for\t\r \u00a024\t\r \u00a0h\t\r \u00a0as\t\r \u00a0described\t\r \u00a0above.\t\r \u00a0An\t\r \u00a0equal\t\r \u00a0number\t\r \u00a0of\t\r \u00a0transfected\t\r \u00a0cells\t\r \u00a0and\t\r \u00a0heavy\t\r \u00a0SILAC-\u00ad\u2010labeled\t\r \u00a0cells\t\r \u00a0were\t\r \u00a0mixed\t\r \u00a0and\t\r \u00a0solubilized\t\r \u00a0in\t\r \u00a0Lysis\t\r \u00a0Buffer\t\r \u00a0(20\t\r \u00a0mM\t\r \u00a0Tris,\t\r \u00a0150\t\r \u00a0mM\t\r \u00a0NaCl,\t\r \u00a01%\t\r \u00a0Nonidet\t\r \u00a0P-\u00ad\u201040,\t\r \u00a01\t\r \u00a0mM\t\r \u00a0sodium\t\r \u00a0orthovanadate,\t\r \u00a010\t\r \u00a0mM\t\r \u00a0sodium\t\r \u00a0pyrophosphate,\t\r \u00a050\t\r \u00a0mM\t\r \u00a0sodium\t\r \u00a0fluoride,\t\r \u00a0and\t\r \u00a0protease\t\r \u00a0inhibitor\t\r \u00a0cocktail\t\r \u00a0(Complete,\t\r \u00a0 \t\r \u00a0\t\r \u00a0 63\t\r \u00a0 Roche\t\r \u00a0Diagnostics)\t\r \u00a0pH\t\r \u00a07.5).\t\r \u00a0The\t\r \u00a0lysate\t\r \u00a0was\t\r \u00a0then\t\r \u00a0centrifuged\t\r \u00a0at\t\r \u00a016,100\t\r \u00a0g\t\r \u00a0for\t\r \u00a010\t\r \u00a0min\t\r \u00a0at\t\r \u00a04\u00baC.\t\r \u00a0Separately,\t\r \u00a025\t\r \u00a0\u03bcL\t\r \u00a0of\t\r \u00a0Protein\t\r \u00a0G\t\r \u00a0SeparoseTM\t\r \u00a04\t\r \u00a0Fast\t\r \u00a0Flow\t\r \u00a0beads\t\r \u00a0(GE\t\r \u00a0Healthcare)\t\r \u00a0were\t\r \u00a0incubated\t\r \u00a0with\t\r \u00a020\t\r \u00a0\u03bcL\t\r \u00a0mouse\t\r \u00a0anti-\u00ad\u2010HA\t\r \u00a0antibody\t\r \u00a0in\t\r \u00a01\t\r \u00a0mL\t\r \u00a0PBS\t\r \u00a0at\t\r \u00a04\u00baC\t\r \u00a0for\t\r \u00a02\t\r \u00a0h.\t\r \u00a0The\t\r \u00a0beads\t\r \u00a0were\t\r \u00a0washed\t\r \u00a0three\t\r \u00a0times\t\r \u00a0with\t\r \u00a0Lysis\t\r \u00a0Buffer\t\r \u00a0and\t\r \u00a0combined\t\r \u00a0with\t\r \u00a0the\t\r \u00a0clarified\t\r \u00a0cell\t\r \u00a0lysate\t\r \u00a0for\t\r \u00a02\t\r \u00a0h\t\r \u00a0at\t\r \u00a04\u00baC.\t\r \u00a0Subsequently,\t\r \u00a0the\t\r \u00a0beads\t\r \u00a0were\t\r \u00a0washed\t\r \u00a0three\t\r \u00a0times\t\r \u00a0with\t\r \u00a0Lysis\t\r \u00a0Buffer\t\r \u00a0and\t\r \u00a0all\t\r \u00a0liquid\t\r \u00a0was\t\r \u00a0removed\t\r \u00a0from\t\r \u00a0the\t\r \u00a0beads\t\r \u00a0after\t\r \u00a0each\t\r \u00a0wash\t\r \u00a0using\t\r \u00a0GELoader\t\r \u00a0Tips\t\r \u00a0(Eppendorf,\t\r \u00a0Westbury,\t\r \u00a0NY).\t\r \u00a0Following\t\r \u00a0the\t\r \u00a0wash,\t\r \u00a0the\t\r \u00a0beads\t\r \u00a0were\t\r \u00a0resuspended\t\r \u00a0in\t\r \u00a020\t\r \u00a0\u03bcL\t\r \u00a0SDS\t\r \u00a0Sample\t\r \u00a0Buffer\t\r \u00a0(50\t\r \u00a0mM\t\r \u00a0Tris,\t\r \u00a02%\t\r \u00a0Sodium\t\r \u00a0dodecyl\t\r \u00a0sulfate,\t\r \u00a010%\t\r \u00a0glycerol),\t\r \u00a0heated\t\r \u00a0to\t\r \u00a099\u00baC\t\r \u00a0for\t\r \u00a01\t\r \u00a0min,\t\r \u00a0and\t\r \u00a0separated\t\r \u00a0on\t\r \u00a0a\t\r \u00a010%\t\r \u00a0acrylamide\t\r \u00a0gel\t\r \u00a0by\t\r \u00a0SDS-\u00ad\u2010PAGE.\t\r \u00a0The\t\r \u00a0lane\t\r \u00a0containing\t\r \u00a0the\t\r \u00a02HA-\u00ad\u2010SopB\t\r \u00a0immunoprecipitation\t\r \u00a0was\t\r \u00a0cut\t\r \u00a0into\t\r \u00a0twelve\t\r \u00a0slices\t\r \u00a0for\t\r \u00a0proteolytic\t\r \u00a0digestion.\t\r \u00a0\t\r \u00a0 2.2.5\t\r \u00a0GST-\u00ad\u2010SopB\t\r \u00a0pull\t\r \u00a0downs\t\r \u00a0Both\t\r \u00a0heavy\t\r \u00a0SILAC\t\r \u00a0and\t\r \u00a0unlabeled\t\r \u00a0HeLa\t\r \u00a0cell\t\r \u00a0lysates\t\r \u00a0were\t\r \u00a0prepared\t\r \u00a0by\t\r \u00a0scraping\t\r \u00a0the\t\r \u00a0cells\t\r \u00a0from\t\r \u00a0six\t\r \u00a0confluent\t\r \u00a010\t\r \u00a0cm\t\r \u00a0plates\t\r \u00a0each.\t\r \u00a0The\t\r \u00a0resulting\t\r \u00a0cell\t\r \u00a0pellets\t\r \u00a0were\t\r \u00a0washed\t\r \u00a0three\t\r \u00a0times\t\r \u00a0with\t\r \u00a0cold\t\r \u00a0PBS\t\r \u00a0and\t\r \u00a0the\t\r \u00a0cells\t\r \u00a0lysed\t\r \u00a0in\t\r \u00a01\t\r \u00a0ml\t\r \u00a0of\t\r \u00a0cold\t\r \u00a0lysis\t\r \u00a0buffer\t\r \u00a0(20\t\r \u00a0mM\t\r \u00a0Tris-\u00ad\u2010Cl,\t\r \u00a050\t\r \u00a0mM\t\r \u00a0NaCl,\t\r \u00a01%\t\r \u00a0NP-\u00ad\u201040,\t\r \u00a03\t\r \u00a0mM\t\r \u00a0MgCl2,\t\r \u00a01\t\r \u00a0mM\t\r \u00a0CaCl2,\t\r \u00a01\t\r \u00a0mM\t\r \u00a0EDTA,\t\r \u00a0pH7.5)\t\r \u00a0containing\t\r \u00a0protease\t\r \u00a0inhibitors\t\r \u00a0(Complete\t\r \u00a0tablets,\t\r \u00a0Roche\t\r \u00a0Diagnostics).\t\r \u00a0The\t\r \u00a0insoluble\t\r \u00a0material\t\r \u00a0was\t\r \u00a0spun\t\r \u00a0down\t\r \u00a0and\t\r \u00a0the\t\r \u00a0resulting\t\r \u00a0soluble\t\r \u00a0fraction\t\r \u00a0was\t\r \u00a0pre-\u00ad\u2010cleared\t\r \u00a0for\t\r \u00a030\t\r \u00a0min\t\r \u00a0at\t\r \u00a04\u00b0C\t\r \u00a0with\t\r \u00a0glutathione-\u00ad\u2010agarose\t\r \u00a0beads\t\r \u00a0containing\t\r \u00a0no\t\r \u00a0bound\t\r \u00a0protein.\t\r \u00a0The\t\r \u00a0heavy\t\r \u00a0SILAC\t\r \u00a0lysate\t\r \u00a0was\t\r \u00a0then\t\r \u00a0incubated\t\r \u00a0with\t\r \u00a050\t\r \u00a0ml\t\r \u00a0of\t\r \u00a0the\t\r \u00a0beads\t\r \u00a0containing\t\r \u00a0bound\t\r \u00a0GST-\u00ad\u2010SopB\t\r \u00a0for\t\r \u00a02\t\r \u00a0hrs\t\r \u00a0at\t\r \u00a04\u00b0C.\t\r \u00a0Similarly,\t\r \u00a0beads\t\r \u00a0containing\t\r \u00a0bound\t\r \u00a0GST\t\r \u00a0only\t\r \u00a0were\t\r \u00a0incubated\t\r \u00a0with\t\r \u00a0the\t\r \u00a0unlabeled\t\r \u00a0HeLa\t\r \u00a0lysate\t\r \u00a0so\t\r \u00a0that\t\r \u00a0equal\t\r \u00a0amounts\t\r \u00a0of\t\r \u00a0GST\t\r \u00a0and\t\r \u00a0GST-\u00ad\u2010SopB\t\r \u00a0were\t\r \u00a0 \t\r \u00a0\t\r \u00a0 64\t\r \u00a0 added\t\r \u00a0to\t\r \u00a0the\t\r \u00a0appropriate\t\r \u00a0lysates.\t\r \u00a0The\t\r \u00a0beads\t\r \u00a0were\t\r \u00a0washed\t\r \u00a0three\t\r \u00a0times\t\r \u00a0with\t\r \u00a0lysis\t\r \u00a0buffer,\t\r \u00a0combined\t\r \u00a0into\t\r \u00a0a\t\r \u00a0single\t\r \u00a0tube\t\r \u00a0and\t\r \u00a0washed\t\r \u00a0another\t\r \u00a0three\t\r \u00a0times\t\r \u00a0with\t\r \u00a0PBS\t\r \u00a0and\t\r \u00a0then\t\r \u00a0diluted\t\r \u00a0to\t\r \u00a0a\t\r \u00a050%\t\r \u00a0slurry\t\r \u00a0in\t\r \u00a0PBS.\t\r \u00a0Two\t\r \u00a0units\t\r \u00a0of\t\r \u00a0thrombin\t\r \u00a0protease\t\r \u00a0(GE\t\r \u00a0Healthcare)\t\r \u00a0were\t\r \u00a0added\t\r \u00a0and\t\r \u00a0the\t\r \u00a0cleavage\t\r \u00a0reaction\t\r \u00a0was\t\r \u00a0allowed\t\r \u00a0to\t\r \u00a0proceed\t\r \u00a0in\t\r \u00a0a\t\r \u00a0shaker\t\r \u00a0for\t\r \u00a016\t\r \u00a0h\t\r \u00a0at\t\r \u00a022\u00b0C,\t\r \u00a0after\t\r \u00a0which\t\r \u00a0an\t\r \u00a0ethanol\/sodium\t\r \u00a0acetate\t\r \u00a0precipitation\t\r \u00a0was\t\r \u00a0performed\t\r \u00a0on\t\r \u00a0the\t\r \u00a0supernatant(467).\t\r \u00a0The\t\r \u00a0protein\t\r \u00a0pellet\t\r \u00a0was\t\r \u00a0solubilized\t\r \u00a0in\t\r \u00a06\t\r \u00a0M\t\r \u00a0urea\/2\t\r \u00a0M\t\r \u00a0thiourea\t\r \u00a0in\t\r \u00a0preparation\t\r \u00a0for\t\r \u00a0doing\t\r \u00a0an\t\r \u00a0in-\u00ad\u2010solution\t\r \u00a0tryptic\t\r \u00a0digest\t\r \u00a0(see\t\r \u00a0below).\t\r \u00a0\t\r \u00a0 2.2.6\t\r \u00a0Mass\t\r \u00a0spectrometry\t\r \u00a0In-\u00ad\u2010gel(468)\t\r \u00a0and\t\r \u00a0in-\u00ad\u2010solution(467)\t\r \u00a0tryptic\t\r \u00a0digestions\t\r \u00a0were\t\r \u00a0performed\t\r \u00a0as\t\r \u00a0previously\t\r \u00a0described,\t\r \u00a0and\t\r \u00a0peptides\t\r \u00a0were\t\r \u00a0acidified\t\r \u00a0in\t\r \u00a01%\t\r \u00a0trifluoracetic\t\r \u00a0acid,\t\r \u00a00.5%\t\r \u00a0acetic\t\r \u00a0acid,\t\r \u00a0and\t\r \u00a03%\t\r \u00a0acetonitrile\t\r \u00a0(Sample\t\r \u00a0Buffer).\t\r \u00a0Protein\t\r \u00a0was\t\r \u00a0concentrated\t\r \u00a0and\t\r \u00a0purified\t\r \u00a0on\t\r \u00a0STop\t\r \u00a0And\t\r \u00a0Go\t\r \u00a0Extraction\t\r \u00a0(STAGE)\t\r \u00a0tips(469),\t\r \u00a0eluted\t\r \u00a0in\t\r \u00a080%\t\r \u00a0acetonitrile,\t\r \u00a00.5%\t\r \u00a0acetic\t\r \u00a0acid,\t\r \u00a0dried\t\r \u00a0in\t\r \u00a0a\t\r \u00a0vacuum\t\r \u00a0concentrator,\t\r \u00a0and\t\r \u00a0resuspended\t\r \u00a0in\t\r \u00a03.2\t\r \u00a0\u03bcL\t\r \u00a0Sample\t\r \u00a0Buffer\t\r \u00a0for\t\r \u00a0analysis.\t\r \u00a0For\t\r \u00a0liquid\t\r \u00a0chromatography-\u00ad\u2010tandem\t\r \u00a0mass\t\r \u00a0spectrometry,\t\r \u00a0samples\t\r \u00a0were\t\r \u00a0analyzed\t\r \u00a0on\t\r \u00a0a\t\r \u00a0LTQ-\u00ad\u2010Orbitrap\t\r \u00a0(Thermo\t\r \u00a0Fisher\t\r \u00a0Scientific,\t\r \u00a0Bremen,\t\r \u00a0Germany)\t\r \u00a0coupled\t\r \u00a0on-\u00ad\u2010line\t\r \u00a0to\t\r \u00a0an\t\r \u00a0Agilent\t\r \u00a01100\t\r \u00a0Series\t\r \u00a0nanoflow\t\r \u00a0HPLC\t\r \u00a0instrument\t\r \u00a0using\t\r \u00a0a\t\r \u00a0nanospray\t\r \u00a0ionization\t\r \u00a0source\t\r \u00a0(Proxeon\t\r \u00a0Biosystems)\t\r \u00a0as\t\r \u00a0previously\t\r \u00a0described(304).\t\r \u00a0Buffer\t\r \u00a0A\t\r \u00a0consisted\t\r \u00a0of\t\r \u00a00.5%\t\r \u00a0acetic\t\r \u00a0acid,\t\r \u00a0and\t\r \u00a0buffer\t\r \u00a0B\t\r \u00a0consisted\t\r \u00a0of\t\r \u00a00.5%\t\r \u00a0acetic\t\r \u00a0acid\t\r \u00a0and\t\r \u00a080%\t\r \u00a0acetonitrile.\t\r \u00a0LC\t\r \u00a0gradients\t\r \u00a0were\t\r \u00a0run\t\r \u00a0from\t\r \u00a06%\t\r \u00a0buffer\t\r \u00a0B\t\r \u00a0to\t\r \u00a030%\t\r \u00a0buffer\t\r \u00a0B\t\r \u00a0during\t\r \u00a0the\t\r \u00a0first\t\r \u00a060\t\r \u00a0min,\t\r \u00a0then\t\r \u00a030%\t\r \u00a0buffer\t\r \u00a0B\t\r \u00a0to\t\r \u00a080%\t\r \u00a0buffer\t\r \u00a0B\t\r \u00a0in\t\r \u00a0the\t\r \u00a0next\t\r \u00a010\t\r \u00a0min.\t\r \u00a0The\t\r \u00a0gradients\t\r \u00a0were\t\r \u00a0then\t\r \u00a0held\t\r \u00a0at\t\r \u00a080%\t\r \u00a0buffer\t\r \u00a0B\t\r \u00a0for\t\r \u00a05\t\r \u00a0min,\t\r \u00a0and\t\r \u00a0dropped\t\r \u00a0to\t\r \u00a06%\t\r \u00a0buffer\t\r \u00a0B\t\r \u00a0for\t\r \u00a015\t\r \u00a0min\t\r \u00a0to\t\r \u00a0 \t\r \u00a0\t\r \u00a0 65\t\r \u00a0 recondition\t\r \u00a0the\t\r \u00a0column.\t\r \u00a0The\t\r \u00a0LTQ-\u00ad\u2010Orbitrap\t\r \u00a0was\t\r \u00a0set\t\r \u00a0at\t\r \u00a060,000\t\r \u00a0resolution\t\r \u00a0and\t\r \u00a0to\t\r \u00a0acquire\t\r \u00a0a\t\r \u00a0full-\u00ad\u2010range\t\r \u00a0scan\t\r \u00a0from\t\r \u00a0350-\u00ad\u20101500\t\r \u00a0m\/z,\t\r \u00a0and\t\r \u00a0simultaneously\t\r \u00a0fragment\t\r \u00a0the\t\r \u00a0top\t\r \u00a0five\t\r \u00a0peptide\t\r \u00a0ions\t\r \u00a0in\t\r \u00a0each\t\r \u00a0cycle\t\r \u00a0within\t\r \u00a0the\t\r \u00a0LTQ.\t\r \u00a0Monoisotopic\t\r \u00a0peaks\t\r \u00a0and\t\r \u00a0charge\t\r \u00a0states\t\r \u00a0in\t\r \u00a0the\t\r \u00a0data\t\r \u00a0were\t\r \u00a0extracted\t\r \u00a0and\t\r \u00a0corrected\t\r \u00a0using\t\r \u00a0DTA\t\r \u00a0Supercharge\t\r \u00a0(http:\/\/msquant.sourceforge.net\/),\t\r \u00a0and\t\r \u00a0peak\t\r \u00a0lists\t\r \u00a0were\t\r \u00a0searched\t\r \u00a0against\t\r \u00a0a\t\r \u00a0database\t\r \u00a0containing\t\r \u00a0all\t\r \u00a0human\t\r \u00a0protein\t\r \u00a0sequences\t\r \u00a0in\t\r \u00a0the\t\r \u00a0International\t\r \u00a0Proteome\t\r \u00a0Index\t\r \u00a0plus\t\r \u00a0the\t\r \u00a0sequences\t\r \u00a0of\t\r \u00a0all\t\r \u00a0Salmonella\t\r \u00a0strain\t\r \u00a0SL1344\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0and\t\r \u00a02\t\r \u00a0effectors\t\r \u00a0(60,137\t\r \u00a0sequences)\t\r \u00a0using\t\r \u00a0Mascot\t\r \u00a0(v2.1,\t\r \u00a0Matrix\t\r \u00a0Science).\t\r \u00a0Search\t\r \u00a0parameters\t\r \u00a0included\t\r \u00a0one\t\r \u00a0missed\t\r \u00a0cleavage\t\r \u00a0site,\t\r \u00a0cysteine\t\r \u00a0carbamidomethyl\t\r \u00a0fixed\t\r \u00a0modification,\t\r \u00a0and\t\r \u00a0variable\t\r \u00a0modifications\t\r \u00a0including\t\r \u00a0methionine\t\r \u00a0oxidation\t\r \u00a0and\t\r \u00a0quadruply-\u00ad\u2010deuterated\t\r \u00a0lysine\t\r \u00a0and\t\r \u00a0 13C6-\u00ad\u2010arginine\t\r \u00a0for\t\r 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\u00a0described\t\r \u00a0except\t\r \u00a0that\t\r \u00a0Protein\t\r \u00a0A\t\r \u00a0SeparoseTM\t\r \u00a04\t\r \u00a0Fast\t\r \u00a0Flow\t\r \u00a0beads\t\r \u00a0(GE\t\r \u00a0 \t\r \u00a0\t\r \u00a0 66\t\r \u00a0 Healthcare)\t\r \u00a0and\t\r \u00a0a\t\r \u00a0rabbit\t\r \u00a0antibody\t\r \u00a0to\t\r \u00a0SopB\t\r \u00a0were\t\r \u00a0used.\t\r \u00a0Proteins\t\r \u00a0resolved\t\r \u00a0by\t\r \u00a010%\t\r \u00a0SDS-\u00ad\u2010PAGE\t\r \u00a0were\t\r \u00a0transferred\t\r \u00a0to\t\r \u00a0nitrocellulose\t\r \u00a0and\t\r \u00a0blocked\t\r \u00a0in\t\r \u00a0PBS-\u00ad\u2010T\t\r \u00a0(PBS\t\r \u00a0and\t\r \u00a00.1%\t\r \u00a0Tween20)\t\r \u00a0with\t\r \u00a05%\t\r \u00a0milk\t\r \u00a0powder\t\r \u00a0for\t\r \u00a030\t\r \u00a0min.\t\r \u00a0Blots\t\r \u00a0were\t\r \u00a0incubated\t\r \u00a0with\t\r \u00a0a\t\r \u00a0primary\t\r \u00a0mouse\t\r \u00a0antibody\t\r \u00a0to\t\r \u00a0Cdc42\t\r \u00a0(BD\t\r \u00a0Transduction\t\r \u00a0Laboratories)\t\r \u00a0at\t\r \u00a0a\t\r \u00a0dilution\t\r \u00a0of\t\r \u00a01:1000\t\r \u00a0in\t\r \u00a0PBS-\u00ad\u2010T\t\r \u00a0with\t\r \u00a05%\t\r \u00a0milk\t\r \u00a0powder\t\r \u00a0for\t\r \u00a01.5\t\r \u00a0h\t\r \u00a0at\t\r \u00a0room\t\r \u00a0temperature\t\r \u00a0(RT),\t\r \u00a0and\t\r \u00a0horseradish\t\r \u00a0peroxidase-\u00ad\u2010conjugated\t\r \u00a0anti-\u00ad\u2010mouse\t\r \u00a0secondary\t\r \u00a0antibody\t\r \u00a0(Bio-\u00ad\u2010Rad)\t\r \u00a0at\t\r \u00a0a\t\r \u00a0dilution\t\r \u00a0of\t\r \u00a01:2000\t\r \u00a0in\t\r \u00a0PBS-\u00ad\u2010T\t\r \u00a0with\t\r \u00a05%\t\r \u00a0milk\t\r \u00a0powder\t\r \u00a0for\t\r \u00a01\t\r \u00a0h\t\r \u00a0at\t\r \u00a0RT.\t\r \u00a0The\t\r \u00a0SuperSignal\t\r \u00a0West\t\r \u00a0Pico\t\r \u00a0Chemiluminescent\t\r \u00a0(Pierce)\t\r \u00a0detection\t\r \u00a0system\t\r \u00a0was\t\r \u00a0used\t\r \u00a0according\t\r \u00a0to\t\r \u00a0the\t\r \u00a0manufacturer\u2019s\t\r \u00a0directions.\t\r \u00a0\t\r \u00a0 2.2.8\t\r \u00a0Immunoflourescence\t\r \u00a0HeLa\t\r \u00a0cells\t\r \u00a0were\t\r \u00a0grown\t\r \u00a0on\t\r \u00a012\t\r \u00a0mm\t\r \u00a0diameter\t\r \u00a0glass\t\r \u00a0coverslips\t\r \u00a0and\t\r \u00a0transfected\t\r \u00a0as\t\r \u00a0described\t\r \u00a0above.\t\r \u00a0Cells\t\r \u00a0were\t\r \u00a0fixed\t\r \u00a0with\t\r \u00a04%\t\r \u00a0formaldehyde\t\r \u00a0for\t\r \u00a010\t\r \u00a0min\t\r \u00a0at\t\r \u00a04\u00baC,\t\r \u00a0washed\t\r \u00a0in\t\r \u00a0PBS,\t\r \u00a0permeabilized\t\r \u00a0in\t\r \u00a0PBS\t\r \u00a0with\t\r \u00a00.1%\t\r \u00a0Triton-\u00ad\u2010X-\u00ad\u2010100\t\r \u00a0(v\/v)\t\r \u00a0and\t\r \u00a050\t\r \u00a0mM\t\r \u00a0NH4Cl\t\r \u00a0for\t\r \u00a020\t\r \u00a0min\t\r \u00a0at\t\r \u00a0RT\t\r \u00a0and\t\r \u00a0blocked\t\r \u00a0in\t\r \u00a0PBS\t\r \u00a0with\t\r \u00a03%\t\r \u00a0milk\t\r \u00a0powder\t\r \u00a0for\t\r \u00a030\t\r \u00a0min\t\r \u00a0at\t\r \u00a0RT.\t\r \u00a0Primary\t\r \u00a0anti-\u00ad\u2010Myc\t\r \u00a0antibody\t\r \u00a0(9E10,\t\r \u00a0Developmental\t\r \u00a0Studies\t\r \u00a0Hybridoma\t\r \u00a0Bank,\t\r \u00a0Iowa\t\r \u00a0City,\t\r \u00a0IA)\t\r \u00a0was\t\r \u00a0overlaid\t\r \u00a0on\t\r \u00a0coverslips\t\r \u00a0for\t\r \u00a02\t\r \u00a0h\t\r \u00a0at\t\r \u00a0RT\t\r \u00a0at\t\r \u00a0a\t\r \u00a0dilution\t\r \u00a0of\t\r \u00a01:100\t\r \u00a0in\t\r \u00a0PBS\t\r \u00a0with\t\r \u00a03%\t\r \u00a0milk\t\r \u00a0powder.\t\r \u00a0Cells\t\r \u00a0were\t\r \u00a0washed\t\r \u00a0with\t\r \u00a0PBS\t\r \u00a0and\t\r \u00a0a\t\r \u00a0secondary\t\r \u00a0Cy3-\u00ad\u2010labeled\t\r \u00a0goat\t\r \u00a0anti-\u00ad\u2010mouse\t\r \u00a0antibody\t\r \u00a0(Jackson\t\r \u00a0ImmunoResearch\t\r \u00a0Laboratories\t\r \u00a0Inc.)\t\r \u00a0was\t\r \u00a0overlaid\t\r \u00a0on\t\r \u00a0coverslips\t\r \u00a0for\t\r \u00a01\t\r \u00a0h\t\r \u00a0at\t\r \u00a0RT\t\r \u00a0at\t\r \u00a0a\t\r \u00a0dilution\t\r \u00a0of\t\r \u00a01:250\t\r \u00a0in\t\r \u00a0PBS\t\r \u00a0with\t\r \u00a03%\t\r \u00a0milk\t\r \u00a0powder.\t\r \u00a0Slides\t\r \u00a0were\t\r \u00a0mounted\t\r \u00a0onto\t\r \u00a01\t\r \u00a0mm\t\r \u00a0glass\t\r \u00a0slides\t\r \u00a0using\t\r \u00a0Prolong\t\r \u00a0Gold\t\r \u00a0antifade\t\r \u00a0reagent\t\r \u00a0(Invitrogen).\t\r \u00a0Images\t\r \u00a0were\t\r \u00a0acquired\t\r \u00a0on\t\r \u00a0an\t\r \u00a0Olympus\t\r \u00a01X81\t\r \u00a0Microscope,\t\r \u00a0using\t\r \u00a0a\t\r \u00a0100x\t\r \u00a0(NA1.4)\t\r \u00a0objective\t\r \u00a0and\t\r \u00a0a\t\r \u00a0COOLSNAP\t\r \u00a0HQ2\t\r \u00a0camera.\t\r \u00a0Colocalization\t\r \u00a0was\t\r \u00a0determined\t\r \u00a0by\t\r \u00a0 \t\r \u00a0\t\r \u00a0 67\t\r \u00a0 calculating\t\r \u00a0correlation\t\r \u00a0coefficients\t\r \u00a0between\t\r \u00a0channel\t\r \u00a0intensities\t\r \u00a0on\t\r \u00a0deconvolved\t\r \u00a0images\t\r \u00a0using\t\r \u00a0SlideBook4.1\t\r \u00a0software.\t\r \u00a0\t\r \u00a0 2.2.9\t\r \u00a0Myc-\u00ad\u2010Cdc42\t\r \u00a0immunoprecipitations\t\r \u00a0For\t\r \u00a0each\t\r \u00a0condition\t\r \u00a0two\t\r \u00a010\t\r \u00a0cm\t\r \u00a0plates\t\r \u00a0of\t\r \u00a0HEK293\t\r \u00a0cells\t\r \u00a0were\t\r \u00a0co-\u00ad\u2010transfected\t\r \u00a0with\t\r \u00a0Myc-\u00ad\u2010Cdc42\t\r \u00a0and\t\r \u00a0one\t\r \u00a0of\t\r \u00a0the\t\r \u00a0GFP-\u00ad\u2010tagged\t\r \u00a0constructs\t\r \u00a0described\t\r \u00a0above,\t\r \u00a0and\t\r \u00a0protein\t\r \u00a0concentration\t\r \u00a0was\t\r \u00a0normalized\t\r \u00a0between\t\r \u00a0samples\t\r \u00a0using\t\r \u00a0a\t\r \u00a0standard\t\r \u00a0Bradford\t\r \u00a0Assay\t\r \u00a0(Pierce).\t\r \u00a0Immunoprecipitations\t\r \u00a0were\t\r \u00a0performed\t\r \u00a0as\t\r \u00a0described\t\r \u00a0above,\t\r \u00a0and\t\r \u00a0proteins\t\r \u00a0resolved\t\r \u00a0by\t\r \u00a0SDS-\u00ad\u2010PAGE\t\r \u00a0were\t\r \u00a0detected\t\r \u00a0by\t\r \u00a0western\t\r \u00a0blotting\t\r \u00a0as\t\r \u00a0previously\t\r \u00a0described\t\r \u00a0using\t\r \u00a0either\t\r \u00a0a\t\r \u00a0mouse\t\r \u00a0antibody\t\r \u00a0to\t\r \u00a0Myc\t\r \u00a0or\t\r \u00a0a\t\r \u00a0rabbit\t\r \u00a0antibody\t\r \u00a0to\t\r \u00a0GFP\t\r \u00a0(Roche),\t\r \u00a0and\t\r \u00a0a\t\r \u00a0horseradish\t\r \u00a0peroxidase-\u00ad\u2010conjugated\t\r \u00a0anti-\u00ad\u2010rabbit\t\r \u00a0or\t\r \u00a0anti-\u00ad\u2010mouse\t\r \u00a0secondary\t\r \u00a0antibody\t\r \u00a0(Bio-\u00ad\u2010Rad).\t\r \u00a0\t\r \u00a0 2.2.10\t\r \u00a0Cdc42\t\r \u00a0activity\t\r \u00a0assays\t\r \u00a0Four\t\r \u00a010\t\r \u00a0cm\t\r \u00a0plates\t\r \u00a0of\t\r \u00a0HEK293\t\r \u00a0were\t\r \u00a0transfected\t\r \u00a0with\t\r \u00a0Myc-\u00ad\u2010Cdc42\t\r \u00a0as\t\r \u00a0described\t\r \u00a0above.\t\r \u00a0Of\t\r \u00a0these\t\r \u00a0four,\t\r \u00a0one\t\r \u00a0plate\t\r \u00a0was\t\r \u00a0also\t\r \u00a0co-\u00ad\u2010transfected\t\r \u00a0with\t\r \u00a0each\t\r \u00a0of\t\r \u00a0SopB29-\u00ad\u2010116-\u00ad\u2010GFP,\t\r \u00a0SopB29-\u00ad\u2010168-\u00ad\u2010GFP,\t\r \u00a0GFP-\u00ad\u2010SopB\u039428(C462S),\t\r \u00a0and\t\r \u00a0GFP-\u00ad\u2010SopB\t\r \u00a0\u039428,\t\r \u00a0one\t\r \u00a0plate\t\r \u00a0was\t\r \u00a0infected\t\r \u00a0with\t\r \u00a0WT,\t\r \u00a0mid-\u00ad\u2010log\t\r \u00a0phase\t\r \u00a0growth\t\r \u00a0S.\t\r \u00a0Typhimurium\t\r \u00a0at\t\r \u00a0a\t\r \u00a0multiplicity\t\r \u00a0of\t\r \u00a0infection\t\r \u00a0of\t\r \u00a025\t\r \u00a0for\t\r \u00a020\t\r \u00a0min\t\r \u00a0at\t\r \u00a037\u00b0C\t\r \u00a0and\t\r \u00a05%\t\r \u00a0CO2,\t\r \u00a0and\t\r \u00a0one\t\r \u00a0plate\t\r \u00a0was\t\r \u00a0treated\t\r \u00a0with\t\r \u00a0GTP\u03b3S\t\r \u00a0according\t\r \u00a0to\t\r \u00a0the\t\r \u00a0manufacturer\u2019s\t\r \u00a0instructions\t\r \u00a0using\t\r \u00a0the\t\r \u00a0Rac\/cdc42\t\r \u00a0Assay\t\r \u00a0Reagent\t\r \u00a0(Millipore).\t\r \u00a0Cells\t\r \u00a0were\t\r \u00a0harvested,\t\r \u00a0lysed,\t\r \u00a0and\t\r \u00a0assayed\t\r \u00a0according\t\r \u00a0to\t\r \u00a0the\t\r \u00a0manufacturer\u2019s\t\r \u00a0instructions,\t\r \u00a0and\t\r \u00a0protein\t\r \u00a0concentration\t\r \u00a0was\t\r \u00a0normalized\t\r \u00a0between\t\r \u00a0samples\t\r \u00a0using\t\r \u00a0a\t\r \u00a0Bradford\t\r \u00a0Assay\t\r \u00a0 \t\r \u00a0\t\r \u00a0 68\t\r \u00a0 as\t\r \u00a0described\t\r \u00a0above.\t\r \u00a0Myc-\u00ad\u2010Cdc42\t\r \u00a0and\t\r \u00a0GFP\t\r \u00a0tagged\t\r \u00a0SopB\t\r \u00a0constructs\t\r \u00a0were\t\r \u00a0detected\t\r \u00a0by\t\r \u00a0Western\t\r \u00a0blot\t\r \u00a0as\t\r \u00a0described\t\r \u00a0above.\t\r \u00a0\t\r \u00a0 2.3\t\r \u00a0Results\t\r \u00a0 2.3.1\t\r \u00a0Cdc42\t\r \u00a0is\t\r \u00a0the\t\r \u00a0host\t\r \u00a0protein\t\r \u00a0target\t\r \u00a0for\t\r \u00a0SopB\t\r \u00a0Modern\t\r \u00a0mass\t\r \u00a0spectrometers\t\r \u00a0in\t\r \u00a0proteomics\t\r \u00a0are\t\r \u00a0exquisitely\t\r \u00a0sensitive\t\r \u00a0instruments,\t\r \u00a0allowing\t\r \u00a0detection\t\r \u00a0of\t\r \u00a0very\t\r \u00a0low\t\r \u00a0abundance\t\r \u00a0proteins\t\r \u00a0in\t\r \u00a0a\t\r \u00a0sample.\t\r \u00a0This\t\r \u00a0is\t\r \u00a0particularly\t\r \u00a0useful\t\r \u00a0for\t\r \u00a0identifying\t\r \u00a0weak\t\r \u00a0or\t\r \u00a0low\t\r \u00a0affinity\t\r \u00a0interactions\t\r \u00a0between\t\r \u00a0proteins,\t\r \u00a0but\t\r \u00a0the\t\r \u00a0sensitivity\t\r \u00a0is\t\r \u00a0so\t\r \u00a0great\t\r \u00a0that\t\r \u00a0non-\u00ad\u2010specific\t\r \u00a0interactions\t\r \u00a0are\t\r \u00a0more\t\r \u00a0frequently\t\r \u00a0identified\t\r \u00a0and\t\r \u00a0are\t\r \u00a0not\t\r \u00a0always\t\r \u00a0recognizable\t\r \u00a0as\t\r \u00a0such.\t\r \u00a0In\t\r \u00a0order\t\r \u00a0to\t\r \u00a0avoid\t\r \u00a0this\t\r \u00a0pitfall\t\r \u00a0in\t\r \u00a0a\t\r \u00a0search\t\r \u00a0for\t\r \u00a0mammalian\t\r \u00a0host\t\r \u00a0targets\t\r \u00a0of\t\r \u00a0SopB,\t\r \u00a0we\t\r \u00a0used\t\r \u00a0SILAC\t\r \u00a0to\t\r \u00a0distinguish\t\r \u00a0specific\t\r \u00a0binding\t\r \u00a0partners\t\r \u00a0for\t\r \u00a0this\t\r \u00a0effector(464)\t\r \u00a0(Figure\t\r \u00a02.1A).\t\r \u00a0A\t\r \u00a0double\t\r \u00a0HA\t\r \u00a0tag\t\r \u00a0was\t\r \u00a0fused\t\r \u00a0to\t\r \u00a0the\t\r \u00a0N-\u00ad\u2010terminus\t\r \u00a0of\t\r \u00a0SopB\t\r \u00a0for\t\r \u00a0transfection\t\r \u00a0into\t\r \u00a0HEK293\t\r \u00a0cells\t\r \u00a0grown\t\r \u00a0in\t\r \u00a0normal\t\r \u00a0media\t\r \u00a0(see\t\r \u00a0Experimental\t\r \u00a0Procedures).\t\r \u00a0The\t\r \u00a0cells\t\r \u00a0were\t\r \u00a0harvested,\t\r \u00a0mixed\t\r \u00a0with\t\r \u00a0an\t\r \u00a0equal\t\r \u00a0number\t\r \u00a0of\t\r \u00a0D4-\u00ad\u2010Lys\t\r \u00a0and\t\r \u00a013C6-\u00ad\u2010Arg\t\r \u00a0labeled\t\r \u00a0cells,\t\r \u00a0lysed\t\r \u00a0and\t\r \u00a0subjected\t\r \u00a0to\t\r \u00a0immunoprecipitation\t\r \u00a0of\t\r \u00a0the\t\r \u00a02HA\t\r \u00a0epitope.\t\r \u00a0The\t\r \u00a0immune\t\r \u00a0complexes\t\r \u00a0were\t\r \u00a0resolved\t\r \u00a0by\t\r \u00a0SDS-\u00ad\u2010PAGE\t\r \u00a0(Figure\t\r \u00a02.1B),\t\r \u00a0and\t\r \u00a0the\t\r \u00a0entire\t\r \u00a0lanes\t\r \u00a0were\t\r \u00a0excised\t\r \u00a0and\t\r \u00a0divided\t\r \u00a0into\t\r \u00a010\t\r \u00a0to\t\r \u00a015\t\r \u00a0slices,\t\r \u00a0digested\t\r \u00a0with\t\r \u00a0trypsin,\t\r \u00a0and\t\r \u00a0analyzed\t\r \u00a0by\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0(Figure\t\r \u00a02.1C).\t\r \u00a0Since\t\r \u00a0normal\t\r \u00a0abundance\t\r \u00a0(light)\t\r \u00a0isotope-\u00ad\u2010labeled\t\r \u00a0cells\t\r \u00a0were\t\r \u00a0transfected,\t\r \u00a0specific\t\r \u00a0SopB\t\r \u00a0binding\t\r \u00a0partners\t\r \u00a0are\t\r \u00a0expected\t\r \u00a0to\t\r \u00a0generate\t\r \u00a0high\t\r \u00a0light:heavy\t\r \u00a0ratios\t\r \u00a0(Figure\t\r \u00a02.1A).\t\r \u00a0The\t\r \u00a0small\t\r \u00a0G-\u00ad\u2010protein\t\r \u00a0Cdc42\t\r \u00a0stood\t\r \u00a0out\t\r \u00a0very\t\r \u00a0clearly\t\r \u00a0as\t\r \u00a0interacting\t\r \u00a0specifically\t\r \u00a0with\t\r \u00a0SopB\t\r \u00a0(Figure\t\r \u00a02.1D),\t\r \u00a0whereas\t\r \u00a0proteins\t\r \u00a0such\t\r \u00a0as\t\r \u00a0eukaryotic\t\r \u00a0initiation\t\r \u00a0factor\t\r \u00a04A-\u00ad\u20101\t\r \u00a0had\t\r \u00a0a\t\r \u00a0ratio\t\r \u00a0near\t\r \u00a01.0\t\r \u00a0(Figure\t\r \u00a02.1E)\t\r \u00a0as\t\r \u00a0predicted\t\r \u00a0for\t\r \u00a0non-\u00ad\u2010specific\t\r \u00a0interactors.\t\r \u00a0As\t\r \u00a0 \t\r \u00a0\t\r \u00a0 69\t\r \u00a0 expected,\t\r \u00a0SopB\t\r \u00a0itself\t\r \u00a0had\t\r \u00a0a\t\r \u00a0very\t\r \u00a0high\t\r \u00a0ratio\t\r \u00a0since\t\r \u00a0it\t\r \u00a0was\t\r \u00a0only\t\r \u00a0expressed\t\r \u00a0in\t\r \u00a0the\t\r \u00a0unlabeled\t\r \u00a0cells.\t\r \u00a0The\t\r \u00a0results\t\r \u00a0of\t\r \u00a0three\t\r \u00a0replicates\t\r \u00a0of\t\r \u00a0this\t\r \u00a0experiment\t\r \u00a0are\t\r \u00a0summarized\t\r \u00a0in\t\r \u00a0Table\t\r \u00a02.1,\t\r \u00a0where\t\r \u00a0proteins\t\r \u00a0with\t\r \u00a0quantitative\t\r \u00a0ratios\t\r \u00a0greater\t\r \u00a0than\t\r \u00a0or\t\r \u00a0equal\t\r \u00a0to\t\r \u00a03.0\t\r \u00a0were\t\r \u00a0considered\t\r \u00a0potential\t\r \u00a0binding\t\r \u00a0partners.\t\r \u00a0The\t\r \u00a0average\t\r \u00a0coefficient\t\r \u00a0of\t\r \u00a0variation\t\r \u00a0(!CV)\t\r \u00a0of\t\r \u00a0all\t\r \u00a0SILAC\t\r \u00a0ratios\t\r \u00a0measured\t\r \u00a0in\t\r \u00a0these\t\r \u00a0experiments\t\r \u00a0was\t\r \u00a029%\t\r \u00a0so\t\r \u00a0a\t\r \u00a0ratio\t\r \u00a0of\t\r \u00a03.0\t\r \u00a0represents\t\r \u00a0~8x\t\r \u00a0!CV,\t\r \u00a0well\t\r \u00a0beyond\t\r \u00a0statistical\t\r \u00a0significance.\t\r \u00a0Intriguingly,\t\r \u00a0the\t\r \u00a0product\t\r \u00a0from\t\r \u00a0one\t\r \u00a0of\t\r \u00a0the\t\r \u00a0four\t\r \u00a0ubiquitin\t\r \u00a0genes\t\r \u00a0in\t\r \u00a0humans\t\r \u00a0also\t\r \u00a0demonstrated\t\r \u00a0a\t\r \u00a0very\t\r \u00a0high\t\r \u00a0ratio.\t\r \u00a0The\t\r \u00a0washing\t\r \u00a0conditions\t\r \u00a0for\t\r \u00a0this\t\r \u00a0experiment\t\r \u00a0were\t\r \u00a0kept\t\r \u00a0relatively\t\r \u00a0mild\t\r \u00a0to\t\r \u00a0preserve\t\r \u00a0potential\t\r \u00a0weak\t\r \u00a0or\t\r \u00a0low\t\r \u00a0affinity\t\r \u00a0interactions,\t\r \u00a0yet\t\r \u00a0of\t\r \u00a0the\t\r \u00a0over\t\r \u00a0300\t\r \u00a0proteins\t\r \u00a0identified,\t\r \u00a0these\t\r \u00a0three\t\r \u00a0polypeptides\t\r \u00a0stood\t\r \u00a0out\t\r \u00a0very\t\r \u00a0clearly.\t\r \u00a0 \t\r \u00a0\t\r \u00a0 70\t\r \u00a0 Figure\t\r \u00a02.1\t\r \u00a0SILAC\t\r \u00a02HA-\u00ad\u2010SopB\t\r \u00a0immunoprecipitations\t\r \u00a0 \t\r \u00a0\t\r \u00a0A)\t\r \u00a0To\t\r \u00a0identify\t\r \u00a0SopB-\u00ad\u2010binding\t\r \u00a0partners,\t\r \u00a0cells\t\r \u00a0grown\t\r \u00a0in\t\r \u00a0normal\t\r \u00a0media\t\r \u00a0were\t\r \u00a0transfected\t\r \u00a0with\t\r \u00a02HA-\u00ad\u2010SopB,\t\r \u00a0harvested,\t\r \u00a0mixed\t\r \u00a0with\t\r \u00a0an\t\r \u00a0equal\t\r \u00a0number\t\r \u00a0of\t\r \u00a0D4-\u00ad\u2010Lys\t\r \u00a0and\t\r \u00a013C6-\u00ad\u2010Arg\t\r \u00a0labeled\t\r \u00a0cells,\t\r \u00a0lysed,\t\r \u00a0and\t\r \u00a0analyzed\t\r \u00a0by\t\r \u00a0mass\t\r \u00a0spectrometry\t\r \u00a0(see\t\r \u00a0Experimental\t\r \u00a0Procedures).\t\r \u00a0Specific\t\r \u00a0binding\t\r \u00a0partners\t\r \u00a0are\t\r \u00a0identified\t\r \u00a0primarily\t\r \u00a0by\t\r \u00a0normal\t\r \u00a0isotopic\t\r \u00a0abundance\t\r \u00a0peptides,\t\r \u00a0and\t\r \u00a0non-\u00ad\u2010specific\t\r \u00a0binding\t\r \u00a0partners\t\r \u00a0are\t\r \u00a0identified\t\r \u00a0by\t\r \u00a0an\t\r \u00a0equal\t\r \u00a0ratio\t\r \u00a0of\t\r \u00a0unlabeled\t\r \u00a0and\t\r \u00a0labeled\t\r \u00a0peptide\t\r \u00a0intensities.\t\r \u00a0B)\t\r \u00a010%\t\r \u00a0SDS-\u00ad\u2010PAGE\t\r \u00a0gel\t\r \u00a0of\t\r \u00a0a\t\r \u00a0representative\t\r \u00a02HA-\u00ad\u2010SopB\t\r \u00a0immunoprecipitate\t\r \u00a0(right\t\r \u00a0lane)\t\r \u00a0that\t\r \u00a0was\t\r \u00a0cut\t\r \u00a0into\t\r \u00a0slices,\t\r \u00a0each\t\r \u00a0of\t\r \u00a0which\t\r \u00a0was\t\r \u00a0analyzed\t\r \u00a0by\t\r \u00a0LC-\u00ad\u2010MSn,\t\r \u00a0generating\t\r \u00a0a\t\r \u00a0total\t\r \u00a0ion\t\r \u00a0chromatogram\t\r \u00a0such\t\r \u00a0as\t\r \u00a0that\t\r \u00a0seen\t\r \u00a0in\t\r \u00a0(C).\t\r \u00a0Mass:charge\t\r \u00a0spectra\t\r \u00a0for\t\r \u00a0representative\t\r \u00a0peptides\t\r \u00a0(sequences\t\r \u00a0shown)\t\r \u00a0identified\t\r \u00a0from\t\r \u00a0Cdc42\t\r \u00a0(D)\t\r \u00a0and\t\r \u00a0eukaryotic\t\r \u00a0initiation\t\r \u00a0factor\t\r \u00a04AI\t\r \u00a0(E)\t\r \u00a0where\t\r \u00a0normal\t\r \u00a0isotope\t\r \u00a0abundance-\u00ad\u2010labeled\t\r \u00a0peptides\t\r \u00a0are\t\r \u00a0indicated\t\r \u00a0with\t\r \u00a0open\t\r \u00a0triangles\t\r \u00a0and\t\r \u00a0D4-\u00ad\u2010Lys\t\r \u00a0or\t\r \u00a013C6-\u00ad\u2010Arg\t\r \u00a0labeled\t\r \u00a0peptides\t\r \u00a0are\t\r \u00a0indicated\t\r \u00a0by\t\r \u00a0filled\t\r \u00a0triangles.\t\r \u00a0The\t\r \u00a0temporal\t\r \u00a0positions\t\r \u00a0of\t\r \u00a0the\t\r \u00a0two\t\r \u00a0spectra\t\r \u00a0shown\t\r \u00a0in\t\r \u00a0D)\t\r \u00a0and\t\r \u00a0E)\t\r \u00a0are\t\r \u00a0indicated\t\r \u00a0in\t\r \u00a0C),\t\r \u00a0and\t\r \u00a0\u2018w\/i\u2019\t\r \u00a0represents\t\r \u00a0the\t\r \u00a0average\t\r \u00a0ratio\t\r \u00a0of\t\r \u00a0the\t\r \u00a0intensity\t\r \u00a0of\t\r \u00a0the\t\r \u00a0unlabeled\t\r \u00a0and\t\r \u00a0labeled\t\r \u00a0forms\t\r \u00a0of\t\r \u00a0the\t\r \u00a0peptide\t\r \u00a0across\t\r \u00a0the\t\r \u00a0chromatographic\t\r \u00a0peak.\t\r \u00a0 \t\r \u00a0\t\r \u00a0 71\t\r \u00a0 \t\r \u00a0 Table\t\r \u00a0 2.1\t\r \u00a0 Summary\t\r \u00a0 of\t\r \u00a0 the\t\r \u00a0 proteins\t\r \u00a0 identified\t\r \u00a0 by\t\r \u00a0 MS\t\r \u00a0 from\t\r \u00a0 three\t\r \u00a0 replicate\t\r \u00a0 SILAC\t\r \u00a0 2HA-\u00ad\u2010SopB\t\r \u00a0 immunoprecipitation\t\r \u00a0experiments\t\r \u00a0\t\r \u00a0 Protein\t\r \u00a0Name1\t\r \u00a0 Sequence\t\r \u00a0coverage2\t\r \u00a0 Peptides3\t\r \u00a0 Ratio4\t\r \u00a0HA-\u00ad\u2010SopB\t\r \u00a0 70\t\r \u00a0 368\t\r \u00a0 >215\t\r \u00a0Isoform\t\r \u00a02\t\r \u00a0of\t\r \u00a0Cell\t\r \u00a0division\t\r \u00a0control\t\r \u00a0protein\t\r \u00a042\t\r \u00a0 39\t\r \u00a0 40\t\r \u00a0 >5\t\r \u00a0Ubiquitin\t\r \u00a0and\t\r \u00a0ribosomal\t\r \u00a0protein\t\r \u00a0S27a\t\r \u00a0 42\t\r \u00a0 91\t\r \u00a0 >5\t\r \u00a0Isoform\t\r \u00a01\t\r \u00a0of\t\r \u00a0Cell\t\r \u00a0division\t\r \u00a0control\t\r \u00a0protein\t\r \u00a042\t\r \u00a0 29\t\r \u00a0 19\t\r \u00a0 >4\t\r \u00a0Eukaryotic\t\r \u00a0initiation\t\r \u00a0factor\t\r \u00a04A-\u00ad\u2010I\t\r \u00a0 22\t\r \u00a0 30\t\r \u00a0 0.94+\/-\u00ad\u20100.23\t\r \u00a040S\t\r \u00a0ribosomal\t\r \u00a0protein\t\r \u00a0S23\t\r \u00a0 31\t\r \u00a0 19\t\r \u00a0 0.89+\/-\u00ad\u20100.23\t\r \u00a0Heat\t\r \u00a0shock\t\r \u00a070kDa\t\r \u00a0protein\t\r \u00a04\t\r \u00a0 13\t\r \u00a0 30\t\r \u00a0 0.88+\/-\u00ad\u20100.29\t\r \u00a014-\u00ad\u20103-\u00ad\u20103\t\r \u00a0zeta\/delta\t\r \u00a0 30\t\r \u00a0 32\t\r \u00a0 0.79+\/-\u00ad\u20100.18\t\r \u00a01Subset\t\r \u00a0of\t\r \u00a0proteins\t\r \u00a0identified\t\r \u00a0in\t\r \u00a0the\t\r \u00a0SopB\t\r \u00a0immunoprecipitation.\t\r \u00a0All\t\r \u00a0proteins\t\r \u00a0with\t\r \u00a0a\t\r \u00a0SILAC\t\r \u00a0ratio\t\r \u00a0greater\t\r \u00a0than\t\r \u00a03\t\r \u00a0are\t\r \u00a0listed\t\r \u00a0along\t\r \u00a0with\t\r \u00a0four\t\r \u00a0representative\t\r \u00a0proteins\t\r \u00a0with\t\r \u00a0ratios\t\r \u00a0near\t\r \u00a01.0\t\r \u00a0from\t\r \u00a0a\t\r \u00a0total\t\r \u00a0of\t\r \u00a0about\t\r \u00a0300\t\r \u00a02Percentage\t\r \u00a0of\t\r \u00a0the\t\r \u00a0entire\t\r \u00a0protein\t\r \u00a0sequence\t\r \u00a0covered\t\r \u00a0by\t\r \u00a0peptides\t\r \u00a0identified\t\r \u00a0by\t\r \u00a0mass\t\r \u00a0spectrometry\t\r \u00a03Number\t\r \u00a0of\t\r \u00a0redundant\t\r \u00a0peptides\t\r \u00a0identified\t\r \u00a04SILAC\t\r \u00a0ratio\t\r \u00a0(light\t\r \u00a0isotope\/heavy\t\r \u00a0isotope)\t\r \u00a0measured\t\r \u00a0for\t\r \u00a0each\t\r \u00a0protein\t\r \u00a05Specific\t\r \u00a0binding\t\r \u00a0partners\t\r \u00a0and\t\r \u00a0the\t\r \u00a0bait\t\r \u00a0itself\t\r \u00a0typically\t\r \u00a0presented\t\r \u00a0with\t\r \u00a0only\t\r \u00a0the\t\r \u00a0unlabeled\t\r \u00a0peptide,\t\r \u00a0leaving\t\r \u00a0an\t\r \u00a0undefined\t\r \u00a0number\t\r \u00a0(division\t\r \u00a0by\t\r \u00a0zero),\t\r \u00a0so\t\r \u00a0ratios\t\r \u00a0are\t\r \u00a0expressed\t\r \u00a0as\t\r \u00a0the\t\r \u00a0minimum\t\r \u00a0signal:noise\t\r \u00a0ratio\t\r 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\u00a0to\t\r \u00a0study\t\r \u00a0Salmonella\t\r \u00a0infection.\t\r \u00a0A\t\r \u00a0lysate\t\r \u00a0of\t\r \u00a0HeLa\t\r \u00a0cells\t\r \u00a0labeled\t\r \u00a0with\t\r \u00a0D4-\u00ad\u2010Lys\t\r \u00a0and\t\r \u00a013C6-\u00ad\u2010Arg\t\r \u00a0was\t\r \u00a0used\t\r \u00a0to\t\r \u00a0fish\t\r \u00a0for\t\r \u00a0proteins\t\r \u00a0binding\t\r \u00a0to\t\r \u00a0SopB,\t\r \u00a0and\t\r \u00a0the\t\r \u00a0resulting\t\r \u00a0complexes\t\r \u00a0were\t\r \u00a0analyzed\t\r \u00a0by\t\r \u00a0MS\t\r \u00a0(see\t\r \u00a0Experimental\t\r \u00a0Procedures).\t\r \u00a0Since\t\r \u00a0GST-\u00ad\u2010SopB\t\r \u00a0was\t\r \u00a0added\t\r \u00a0to\t\r \u00a0heavy\t\r \u00a0isotope-\u00ad\u2010labeled\t\r \u00a0cells,\t\r \u00a0proteins\t\r \u00a0binding\t\r \u00a0specifically\t\r \u00a0to\t\r \u00a0SopB\t\r \u00a0should\t\r \u00a0be\t\r \u00a0present\t\r \u00a0primarily\t\r \u00a0in\t\r \u00a0the\t\r \u00a0heavy\t\r \u00a0form,\t\r \u00a0opposite\t\r \u00a0to\t\r \u00a0the\t\r \u00a0labeling\t\r \u00a0scheme\t\r \u00a0used\t\r \u00a0in\t\r \u00a0the\t\r \u00a0immunoprecipitation\t\r \u00a0experiments\t\r \u00a0discussed\t\r \u00a0above\t\r \u00a0(Figure\t\r \u00a02.1).\t\r \u00a0As\t\r \u00a0with\t\r \u00a0the\t\r \u00a0immunoprecipitation\t\r \u00a0experiments,\t\r \u00a0Cdc42\t\r \u00a0was\t\r \u00a0identified\t\r \u00a0to\t\r \u00a0interact\t\r \u00a0specifically\t\r \u00a0with\t\r \u00a0SopB\t\r \u00a0(Figure\t\r \u00a02.3B).\t\r \u00a0Reversal\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SILAC\t\r \u00a0labeling\t\r \u00a0scheme\t\r \u00a0in\t\r \u00a0these\t\r \u00a0experiments\t\r \u00a0had\t\r \u00a0no\t\r \u00a0impact\t\r \u00a0on\t\r \u00a0Cdc42\t\r \u00a0binding\t\r \u00a0(data\t\r \u00a0not\t\r \u00a0shown).\t\r \u00a0\t\r \u00a0 2.3.3\t\r \u00a0Cdc42\t\r \u00a0is\t\r \u00a0a\t\r \u00a0host\t\r \u00a0target\t\r \u00a0of\t\r \u00a0bacterially\t\r \u00a0delivered\t\r \u00a0SopB\t\r \u00a0We\t\r \u00a0originally\t\r \u00a0identified\t\r \u00a0the\t\r \u00a0SopB-\u00ad\u2010Cdc42\t\r \u00a0interaction\t\r \u00a0in\t\r \u00a0a\t\r \u00a0transfection\t\r \u00a0system,\t\r \u00a0so\t\r \u00a0to\t\r \u00a0confirm\t\r \u00a0that\t\r \u00a0SopB\t\r \u00a0targets\t\r \u00a0Cdc42\t\r \u00a0in\t\r \u00a0a\t\r \u00a0more\t\r \u00a0physiologically\t\r \u00a0relevant\t\r \u00a0system,\t\r \u00a0a\t\r \u00a0SopB\t\r \u00a0antibody\t\r \u00a0was\t\r \u00a0used\t\r \u00a0to\t\r \u00a0immunoprecipitate\t\r \u00a0SopB\t\r \u00a0following\t\r \u00a0its\t\r \u00a0delivery\t\r \u00a0into\t\r \u00a0Raw264.7\t\r \u00a0cells\t\r \u00a0by\t\r \u00a0both\t\r \u00a0wild\t\r \u00a0type\t\r \u00a0and\t\r \u00a0\u0394SopB\t\r \u00a0S.\t\r \u00a0Typhimurium.\t\r \u00a0As\t\r \u00a0seen\t\r \u00a0in\t\r \u00a0Figure\t\r \u00a02.3C,\t\r \u00a0a\t\r \u00a0small\t\r \u00a0fraction\t\r \u00a0of\t\r \u00a0the\t\r \u00a0total\t\r \u00a0Cdc42\t\r \u00a0was\t\r \u00a0very\t\r \u00a0clearly\t\r \u00a0identified\t\r \u00a0in\t\r \u00a0the\t\r \u00a0SopB\t\r 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\u00a0Description\t\r \u00a0of\t\r \u00a02HA-\u00ad\u2010SopB\t\r \u00a0and\t\r \u00a0SopB-\u00ad\u2010GFP\t\r \u00a0and\t\r \u00a0GFP-\u00ad\u2010SopB\t\r \u00a0deletion\t\r \u00a0constructs\t\r \u00a0 \t\r \u00a0Schematic\t\r \u00a0diagram\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SopB-\u00ad\u2010GFP\t\r \u00a0deletion\t\r \u00a0constructs\t\r \u00a0used.\t\r \u00a0C462S\t\r \u00a0represents\t\r \u00a0a\t\r \u00a0point\t\r \u00a0mutation\t\r \u00a0previously\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0render\t\r \u00a0SopB\t\r \u00a0catalytically\t\r \u00a0inactive(458).\t\r \u00a0\t\r \u00a0 \t\r \u00a0\t\r \u00a0 77\t\r \u00a0 Figure\t\r \u00a02.5\t\r \u00a0Colocalization\t\r \u00a0of\t\r \u00a0SopB\t\r \u00a0with\t\r \u00a0Cdc42\t\r \u00a0 \t\r \u00a0HeLa\t\r \u00a0cells\t\r \u00a0were\t\r \u00a0fixed\t\r \u00a0after\t\r \u00a0a\t\r \u00a020\t\r \u00a0h\t\r \u00a0transient\t\r \u00a0cotransfection\t\r \u00a0with\t\r \u00a0GFP\t\r \u00a0and\t\r \u00a0Myc-\u00ad\u2010tagged\t\r 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\u00a0Figure\t\r \u00a02.6,\t\r \u00a0this\t\r \u00a0was\t\r \u00a0done\t\r \u00a0for\t\r \u00a0all\t\r \u00a0seven\t\r \u00a0SopB-\u00ad\u2010GFP\t\r \u00a0deletions,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0a\t\r \u00a0control\t\r \u00a0construct,\t\r \u00a0GFP-\u00ad\u20102FYVE.\t\r \u00a0Control\t\r \u00a0blots\t\r \u00a0were\t\r \u00a0also\t\r \u00a0run\t\r \u00a0to\t\r \u00a0detect\t\r \u00a0expression\t\r \u00a0of\t\r \u00a0each\t\r \u00a0construct,\t\r \u00a0and\t\r \u00a0the\t\r \u00a0results\t\r \u00a0were\t\r \u00a0consistent\t\r \u00a0between\t\r \u00a0three\t\r \u00a0repetitions\t\r \u00a0of\t\r \u00a0the\t\r \u00a0experiment.\t\r \u00a0Recently,\t\r \u00a0the\t\r \u00a0region\t\r \u00a0of\t\r \u00a0SopB\t\r \u00a0between\t\r \u00a0residues\t\r \u00a0118\t\r \u00a0and\t\r \u00a0142\t\r \u00a0was\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0be\t\r \u00a0essential\t\r \u00a0for\t\r \u00a0pulling\t\r \u00a0down\t\r \u00a0Cdc42\t\r \u00a0in\t\r \u00a0Saccharomyces\t\r \u00a0 cerevisiae(462).\t\r \u00a0Consistent\t\r \u00a0with\t\r \u00a0these\t\r \u00a0results,\t\r \u00a0the\t\r \u00a0C-\u00ad\u2010terminal\t\r \u00a0deletion\t\r \u00a0series\t\r \u00a0down\t\r \u00a0to\t\r \u00a0SopB29-\u00ad\u2010168-\u00ad\u2010GFP\t\r \u00a0bound\t\r \u00a0Cdc42,\t\r \u00a0but\t\r \u00a0SopB29-\u00ad\u2010116-\u00ad\u2010GFP\t\r \u00a0did\t\r \u00a0not.\t\r \u00a0This\t\r \u00a0reciprocal\t\r \u00a0immunoprecipitation\t\r \u00a0provides\t\r \u00a0further\t\r \u00a0confirmation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0interaction,\t\r \u00a0and\t\r \u00a0also\t\r \u00a0defines\t\r \u00a0residues\t\r \u00a0117-\u00ad\u2010168\t\r \u00a0as\t\r \u00a0being\t\r \u00a0required\t\r \u00a0for\t\r \u00a0the\t\r \u00a0interaction\t\r \u00a0in\t\r \u00a0mammalian\t\r \u00a0cells.\t\r \u00a0 \t\r \u00a0\t\r \u00a0 79\t\r \u00a0 Figure\t\r \u00a02.6\t\r \u00a0Residues\t\r \u00a0117-\u00ad\u2010168\t\r \u00a0of\t\r \u00a0SopB\t\r \u00a0are\t\r \u00a0required\t\r \u00a0for\t\r 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\u00a0that\t\r \u00a0the\t\r \u00a0doublets,\t\r \u00a0triplets\t\r \u00a0or\t\r \u00a0quadruplets\t\r \u00a0observed\t\r \u00a0for\t\r \u00a0the\t\r \u00a0five\t\r \u00a0SopB-\u00ad\u2010GFP\t\r \u00a0constructs\t\r \u00a0show\t\r \u00a0an\t\r \u00a0approximately\t\r \u00a08\t\r \u00a0kDa\t\r \u00a0separation.\t\r \u00a0Positions\t\r \u00a0of\t\r \u00a0molecular\t\r \u00a0weight\t\r \u00a0markers\t\r \u00a0in\t\r \u00a0kDa\t\r \u00a0are\t\r \u00a0shown\t\r \u00a0on\t\r \u00a0the\t\r \u00a0left\t\r \u00a0side\t\r \u00a0and\t\r \u00a0antibodies\t\r \u00a0used\t\r \u00a0are\t\r \u00a0shown\t\r \u00a0on\t\r \u00a0the\t\r \u00a0right\t\r \u00a0side.\t\r \u00a0 \t\r \u00a0 2.3.6\t\r \u00a0SopB\t\r \u00a0binding\t\r \u00a0does\t\r \u00a0not\t\r \u00a0activate\t\r \u00a0Cdc42\t\r \u00a0 \t\r \u00a0SopB\t\r \u00a0has\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0activate\t\r \u00a0Rho-\u00ad\u2010like\t\r \u00a0GTPases\t\r \u00a0indirectly\t\r \u00a0through\t\r \u00a0the\t\r \u00a0activation\t\r \u00a0of\t\r \u00a0an\t\r \u00a0endogenous\t\r \u00a0GEF\t\r \u00a0(SGEF)(45),\t\r \u00a0and\t\r \u00a0we\t\r \u00a0show\t\r \u00a0here\t\r \u00a0that\t\r \u00a0it\t\r \u00a0also\t\r \u00a0binds\t\r \u00a0Cdc42\t\r \u00a0directly.\t\r \u00a0To\t\r \u00a0determine\t\r \u00a0whether\t\r \u00a0the\t\r \u00a0binding\t\r \u00a0alone,\t\r \u00a0or\t\r \u00a0transfection\t\r \u00a0with\t\r \u00a0full\t\r \u00a0length\t\r \u00a0active\t\r \u00a0SopB\t\r \u00a0is\t\r \u00a0sufficient\t\r \u00a0to\t\r \u00a0alter\t\r \u00a0the\t\r \u00a0activation\t\r \u00a0state\t\r \u00a0of\t\r \u00a0Cdc42,\t\r \u00a0the\t\r \u00a0GST-\u00ad\u2010fused\t\r \u00a0p21-\u00ad\u2010binding\t\r \u00a0domain\t\r \u00a0of\t\r \u00a0human\t\r \u00a0PAK1\t\r \u00a0was\t\r \u00a0used\t\r \u00a0to\t\r \u00a0pull-\u00ad\u2010down\t\r \u00a0active\t\r \u00a0Cdc42\t\r \u00a0from\t\r \u00a0a\t\r \u00a0HEK293\t\r \u00a0lysate\t\r \u00a0(see\t\r \u00a0Experimental\t\r \u00a0Procedures).\t\r 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\u00a0by\t\r \u00a0SDS-\u00ad\u2010PAGE\t\r \u00a0and\t\r \u00a0Western\t\r \u00a0blotted\t\r \u00a0with\t\r \u00a0anti-\u00ad\u2010Myc\t\r \u00a0(top\t\r \u00a0panel).\t\r \u00a0Expression\t\r \u00a0levels\t\r \u00a0of\t\r \u00a0the\t\r \u00a0GFP-\u00ad\u2010constructs\t\r \u00a0(middle\t\r \u00a0panel)\t\r \u00a0and\t\r \u00a0Myc-\u00ad\u2010Cdc42\t\r \u00a0(lower\t\r \u00a0panel)\t\r \u00a0in\t\r \u00a0whole\t\r \u00a0cell\t\r \u00a0lysates\t\r \u00a0are\t\r \u00a0shown.\t\r \u00a0The\t\r \u00a0blot\t\r \u00a0shown\t\r \u00a0represents\t\r \u00a0consistent\t\r \u00a0results\t\r \u00a0from\t\r \u00a0three\t\r \u00a0replicates\t\r \u00a0of\t\r \u00a0the\t\r \u00a0described\t\r \u00a0experiment.\t\r \u00a0Molecular\t\r \u00a0weight\t\r \u00a0markers\t\r \u00a0in\t\r \u00a0kDa\t\r \u00a0are\t\r \u00a0shown\t\r \u00a0on\t\r \u00a0the\t\r \u00a0left\t\r \u00a0side\t\r \u00a0and\t\r \u00a0antibodies\t\r \u00a0used\t\r \u00a0are\t\r \u00a0shown\t\r \u00a0on\t\r \u00a0the\t\r \u00a0right\t\r 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\u00a0 to\t\r \u00a0SopB,\t\r \u00a02)\t\r \u00a0that\t\r \u00a0Cdc42\t\r \u00a0was\t\r \u00a0ubiquitylated,\t\r \u00a0or\t\r \u00a03)\t\r \u00a0that\t\r \u00a0SopB\t\r \u00a0was\t\r \u00a0ubiquitylated.\t\r \u00a0Previously\t\r \u00a0we\t\r \u00a0have\t\r \u00a0shown\t\r \u00a0that\t\r \u00a0bacterially\t\r \u00a0delivered\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0transfected\t\r \u00a0SopB\t\r \u00a0is\t\r \u00a0ubiquitylated\t\r \u00a0in\t\r \u00a0host\t\r \u00a0cells(458).\t\r \u00a0Indeed,\t\r \u00a0when\t\r \u00a0we\t\r \u00a0transfected\t\r \u00a0full-\u00ad\u2010length\t\r \u00a0and\t\r \u00a0truncated\t\r \u00a0SopB\t\r \u00a0constructs\t\r \u00a0in\t\r \u00a0the\t\r \u00a0current\t\r \u00a0study\t\r \u00a0two\t\r \u00a0bands\t\r \u00a0of\t\r \u00a0roughly\t\r \u00a0equal\t\r \u00a0intensity\t\r \u00a0separated\t\r \u00a0by\t\r \u00a0~8\t\r \u00a0kDa\t\r \u00a0are\t\r \u00a0seen\t\r \u00a0by\t\r \u00a0Western\t\r \u00a0blot\t\r \u00a0(Figure\t\r \u00a02.6\t\r \u00a0and\t\r \u00a02.8).\t\r \u00a0This\t\r \u00a0prompted\t\r \u00a0us\t\r \u00a0to\t\r \u00a0further\t\r \u00a0analyze\t\r \u00a0the\t\r \u00a0tandem\t\r \u00a0mass\t\r \u00a0spectra\t\r \u00a0acquired\t\r \u00a0here\t\r \u00a0to\t\r \u00a0search\t\r \u00a0for\t\r \u00a0SopB\t\r \u00a0peptides\t\r \u00a0containing\t\r \u00a0the\t\r \u00a0signature\t\r \u00a0double\t\r \u00a0glycine\t\r \u00a0modification\t\r \u00a0on\t\r \u00a0lysines(473).\t\r \u00a0As\t\r \u00a0a\t\r \u00a0result,\t\r \u00a0lysine\t\r \u00a0residues\t\r \u00a019\t\r \u00a0and\t\r \u00a0541\t\r \u00a0were\t\r \u00a0found\t\r \u00a0to\t\r \u00a0be\t\r \u00a0ubiquitylated\t\r \u00a0following\t\r \u00a02HA-\u00ad\u2010SopB\t\r \u00a0transfections\t\r \u00a0in\t\r \u00a0HEK293\t\r \u00a0cells.\t\r \u00a0The\t\r \u00a0peptide\t\r \u00a0containing\t\r \u00a0K541\t\r \u00a0had\t\r \u00a0only\t\r \u00a0one\t\r \u00a0lysine\t\r \u00a0residue\t\r \u00a0so\t\r \u00a0there\t\r \u00a0was\t\r \u00a0no\t\r \u00a0ambiguity\t\r \u00a0in\t\r \u00a0the\t\r \u00a0assignment,\t\r \u00a0but\t\r \u00a0the\t\r \u00a0peptide\t\r \u00a0containing\t\r \u00a0K19\t\r \u00a0contains\t\r \u00a0two\t\r \u00a0lysine\t\r \u00a0residues\t\r \u00a0(TQEAFKSLQK).\t\r \u00a0Trypsin\t\r \u00a0would\t\r \u00a0be\t\r \u00a0unlikely\t\r \u00a0to\t\r \u00a0cleave\t\r \u00a0after\t\r \u00a0K19\t\r \u00a0if\t\r \u00a0it\t\r \u00a0were\t\r \u00a0modified,\t\r \u00a0and\t\r \u00a0the\t\r \u00a0fragment\t\r \u00a0spectra\t\r \u00a0used\t\r \u00a0for\t\r \u00a0the\t\r \u00a0identification\t\r \u00a0of\t\r \u00a0this\t\r \u00a0peptide\t\r \u00a0provided\t\r \u00a0unequivocal\t\r \u00a0assignment\t\r \u00a0of\t\r \u00a0the\t\r \u00a0modification\t\r \u00a0to\t\r \u00a0that\t\r \u00a0residue\t\r \u00a0(Figure\t\r \u00a02.8).\t\r \u00a0The\t\r \u00a0difference\t\r \u00a0between\t\r \u00a0the\t\r \u00a0y4\t\r \u00a0and\t\r \u00a0y5\t\r \u00a0ions\t\r \u00a0corresponded\t\r \u00a0exactly\t\r \u00a0to\t\r \u00a0the\t\r \u00a0sum\t\r \u00a0of\t\r \u00a0the\t\r \u00a0mass\t\r \u00a0of\t\r \u00a0a\t\r \u00a0lysine\t\r \u00a0plus\t\r \u00a0two\t\r \u00a0glycine\t\r \u00a0residues(473).\t\r \u00a0\t\r \u00a0 \t\r \u00a0 \t\r \u00a0\t\r \u00a0 82\t\r \u00a0 Figure\t\r \u00a02.8\t\r \u00a0Ubiquitylation\t\r \u00a0peptides\t\r \u00a0identified\t\r \u00a0in\t\r \u00a02HA-\u00ad\u2010SopB\t\r \u00a0 \t\r \u00a0A)\t\r \u00a0Shown\t\r \u00a0is\t\r \u00a0a\t\r \u00a0fragment\t\r \u00a0ion\t\r \u00a0spectra\t\r \u00a0for\t\r \u00a0the\t\r \u00a0indicated\t\r \u00a0ubiquitylated\t\r \u00a0SopB\t\r \u00a0peptide\t\r \u00a0containing\t\r \u00a0K19.\t\r \u00a0The\t\r \u00a0identified\t\r \u00a0y-\u00ad\u2010\t\r \u00a0and\t\r \u00a0b-\u00ad\u2010series\t\r \u00a0ions\t\r \u00a0are\t\r \u00a0indicated,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0the\t\r \u00a0mass-\u00ad\u2010to-\u00ad\u2010charge\t\r \u00a0ratios\t\r \u00a0of\t\r \u00a0the\t\r \u00a0abundant\t\r \u00a0y-\u00ad\u2010ions\t\r \u00a0and\t\r \u00a0the\t\r \u00a0difference\t\r \u00a0between\t\r \u00a0y4\t\r \u00a0and\t\r \u00a0y5\t\r \u00a0corresponding\t\r \u00a0to\t\r \u00a0a\t\r \u00a0lysine\t\r \u00a0with\t\r \u00a0two\t\r \u00a0glycines.\t\r \u00a0B)\t\r \u00a0Expression\t\r \u00a0of\t\r \u00a02HA-\u00ad\u2010SopB.\t\r \u00a0Lanes\t\r \u00a0showing\t\r \u00a02HA\t\r \u00a0and\t\r \u00a02HA-\u00ad\u2010SopB\t\r \u00a0expression\t\r \u00a0in\t\r \u00a0HEK293\t\r \u00a0cells\t\r \u00a0and\t\r \u00a0immune\t\r \u00a0blotted\t\r \u00a0with\t\r \u00a0mouse\t\r \u00a0anti-\u00ad\u2010HA\t\r \u00a0are\t\r \u00a0indicated.\t\r \u00a0Note\t\r \u00a0that\t\r \u00a0the\t\r \u00a0doublet\t\r \u00a0observed\t\r \u00a0for\t\r \u00a02HA-\u00ad\u2010SopB\t\r \u00a0shows\t\r \u00a0approximately\t\r \u00a0an\t\r \u00a08\t\r \u00a0kDa\t\r \u00a0separation.\t\r \u00a0Molecular\t\r \u00a0weight\t\r \u00a0markers\t\r \u00a0in\t\r \u00a0kDa\t\r \u00a0are\t\r \u00a0shown\t\r \u00a0on\t\r \u00a0the\t\r \u00a0left\t\r \u00a0side.\t\r \u00a0 \t\r \u00a0 2.4\t\r \u00a0Discussion\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0enables\t\r \u00a0Salmonella\t\r \u00a0to\t\r \u00a0enter\t\r \u00a0non-\u00ad\u2010phagocytic\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0by\t\r \u00a0manipulating\t\r \u00a0the\t\r \u00a0cytoskeleton\t\r \u00a0in\t\r \u00a0the\t\r \u00a0local\t\r \u00a0area\t\r \u00a0of\t\r \u00a0invasion,\t\r \u00a0and\t\r \u00a0it\t\r \u00a0also\t\r \u00a0induces\t\r \u00a0the\t\r \u00a0initial\t\r \u00a0production\t\r \u00a0of\t\r \u00a0proinflammatory\t\r \u00a0cytokines(45,\t\r \u00a0456,\t\r \u00a0457).\t\r \u00a0This\t\r \u00a0is\t\r \u00a0achieved\t\r \u00a0in\t\r \u00a0part\t\r \u00a0through\t\r \u00a0the\t\r \u00a0action\t\r \u00a0of\t\r \u00a0effectors\t\r \u00a0SopE\/E2,\t\r \u00a0which\t\r \u00a0act\t\r \u00a0as\t\r \u00a0GEFs\t\r \u00a0for\t\r \u00a0Cdc42\t\r \u00a0and\t\r \u00a0Rac1,\t\r \u00a0and\t\r \u00a0SptP\t\r \u00a0which\t\r \u00a0acts\t\r \u00a0as\t\r \u00a0a\t\r \u00a0GAP\t\r \u00a0for\t\r \u00a0Cdc42\t\r \u00a0and\t\r \u00a0Rac1.\t\r \u00a0SopB\t\r \u00a0is\t\r \u00a0also\t\r \u00a0known\t\r \u00a0to\t\r \u00a0exert\t\r \u00a0similar\t\r \u00a0effects\t\r \u00a0through\t\r \u00a0phosphoinositide\t\r \u00a0signaling(45,\t\r \u00a0187),\t\r \u00a0and\t\r \u00a0also\t\r \u00a0via\t\r \u00a0an\t\r \u00a0unknown\t\r \u00a0mechanism\t\r \u00a0involving\t\r \u00a0the\t\r \u00a0amino\t\r \u00a0terminal\t\r \u00a0region\t\r \u00a0of\t\r \u00a0the\t\r \u00a0protein(459).\t\r \u00a0Here\t\r \u00a0we\t\r \u00a0have\t\r \u00a0 \t\r \u00a0\t\r \u00a0 83\t\r \u00a0 used\t\r \u00a0an\t\r \u00a0unbiased\t\r \u00a0and\t\r \u00a0very\t\r \u00a0specific\t\r \u00a0quantitative\t\r \u00a0MS-\u00ad\u2010based\t\r \u00a0approach\t\r \u00a0to\t\r \u00a0identify\t\r \u00a0both\t\r \u00a0isoforms\t\r \u00a0of\t\r \u00a0Cdc42\t\r \u00a0as\t\r \u00a0specific\t\r \u00a0host\t\r \u00a0targets\t\r \u00a0of\t\r \u00a0SopB.\t\r \u00a0This\t\r \u00a0interaction\t\r \u00a0was\t\r \u00a0confirmed\t\r \u00a0by\t\r \u00a0GST\t\r \u00a0pull-\u00ad\u2010down\t\r \u00a0assays\t\r \u00a0using\t\r \u00a0recombinant\t\r \u00a0SopB\t\r \u00a0as\t\r \u00a0bait,\t\r \u00a0by\t\r \u00a0reciprocal\t\r \u00a0immunoprecipitation\t\r \u00a0of\t\r \u00a0Cdc42\t\r \u00a0and\t\r \u00a0subsequent\t\r \u00a0detection\t\r \u00a0of\t\r \u00a0SopB,\t\r \u00a0and\t\r \u00a0also\t\r \u00a0immunoprecipitation\t\r \u00a0of\t\r \u00a0bacterially\t\r \u00a0delivered\t\r \u00a0SopB\t\r \u00a0and\t\r \u00a0subsequent\t\r \u00a0detection\t\r \u00a0of\t\r \u00a0Cdc42.\t\r \u00a0The\t\r \u00a0region\t\r \u00a0of\t\r \u00a0SopB\t\r \u00a0between\t\r \u00a0residues\t\r \u00a0117\t\r \u00a0to\t\r \u00a0168\t\r \u00a0is\t\r \u00a0required\t\r \u00a0for\t\r \u00a0binding,\t\r \u00a0suggesting\t\r \u00a0that\t\r \u00a0the\t\r \u00a0effect\t\r \u00a0of\t\r \u00a0the\t\r \u00a0N-\u00ad\u2010terminal\t\r \u00a0region\t\r \u00a0of\t\r \u00a0SopB\t\r \u00a0on\t\r \u00a0the\t\r \u00a0yeast\t\r \u00a0cytoskeleton(459)\t\r \u00a0and\t\r \u00a0membrane\t\r \u00a0targeting(458)\t\r \u00a0is\t\r \u00a0through\t\r \u00a0an\t\r \u00a0effect\t\r \u00a0on\t\r \u00a0Cdc42.\t\r \u00a0During\t\r \u00a0the\t\r \u00a0preparation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0current\t\r \u00a0work,\t\r \u00a0Rodriguez-\u00ad\u2010Escudero\t\r \u00a0et\t\r \u00a0al.\t\r \u00a0reported\t\r \u00a0that\t\r \u00a0SopB\t\r \u00a0can\t\r \u00a0pull\t\r \u00a0down\t\r \u00a0the\t\r \u00a0yeast\t\r \u00a0homologue\t\r \u00a0of\t\r \u00a0Cdc42(462),\t\r \u00a0so\t\r \u00a0our\t\r \u00a0data\t\r \u00a0extend\t\r \u00a0this\t\r \u00a0interaction\t\r \u00a0to\t\r \u00a0mammalian\t\r \u00a0systems\t\r \u00a0capable\t\r \u00a0of\t\r \u00a0being\t\r \u00a0infected\t\r \u00a0by\t\r \u00a0Salmonella.\t\r \u00a0Both\t\r \u00a0our\t\r \u00a0data\t\r \u00a0and\t\r \u00a0that\t\r \u00a0of\t\r \u00a0Rodriguez-\u00ad\u2010Escudero\t\r \u00a0et\t\r \u00a0al.\t\r \u00a0find\t\r \u00a0no\t\r \u00a0evidence\t\r \u00a0that\t\r \u00a0transfection\t\r \u00a0of\t\r \u00a0SopB\t\r \u00a0activates\t\r \u00a0Cdc42\t\r \u00a0dependent\t\r \u00a0signaling\t\r \u00a0or\t\r \u00a0GTPase\t\r \u00a0activity.\t\r \u00a0In\t\r \u00a0contrast,\t\r \u00a0both\t\r \u00a0findings\t\r \u00a0suggest\t\r \u00a0an\t\r \u00a0inhibitory\t\r \u00a0effect\t\r \u00a0of\t\r \u00a0SopB\t\r \u00a0on\t\r \u00a0Cdc42-\u00ad\u2010dependent\t\r \u00a0signaling(462)\t\r \u00a0or\t\r \u00a0GTPase\t\r \u00a0activity\t\r \u00a0(Figure\t\r \u00a02.7),\t\r \u00a0which\t\r \u00a0maybe\t\r \u00a0be\t\r \u00a0due\t\r \u00a0to\t\r \u00a0SopB\t\r \u00a0sequestering\t\r \u00a0Cdc42\t\r \u00a0and\t\r \u00a0suggests\t\r \u00a0that\t\r \u00a0additional\t\r \u00a0factors\t\r \u00a0(most\t\r \u00a0likely\t\r \u00a0bacterial)\t\r \u00a0are\t\r \u00a0required\t\r \u00a0for\t\r \u00a0the\t\r \u00a0previously\t\r \u00a0reported\t\r \u00a0SopB-\u00ad\u2010dependent\t\r \u00a0Cdc42\t\r \u00a0activation(45).\t\r \u00a0Importantly,\t\r \u00a0we\t\r \u00a0find\t\r \u00a0none\t\r \u00a0of\t\r \u00a0the\t\r \u00a0other\t\r \u00a0very\t\r \u00a0closely\t\r \u00a0related\t\r \u00a0Rho-\u00ad\u2010like\t\r \u00a0GTPases\t\r \u00a0or\t\r \u00a0GEFs\t\r \u00a0known\t\r \u00a0to\t\r \u00a0be\t\r \u00a0involved\t\r \u00a0in\t\r \u00a0 Salmonella\t\r \u00a0invasion\t\r \u00a0such\t\r \u00a0as\t\r \u00a0RhoG\t\r \u00a0and\t\r \u00a0Rac1\t\r \u00a0and\t\r \u00a0SGEF\t\r \u00a0complexed\t\r \u00a0with\t\r \u00a0SopB,\t\r \u00a0suggesting\t\r \u00a0that\t\r \u00a0the\t\r \u00a0interaction\t\r \u00a0is\t\r \u00a0specific\t\r \u00a0for\t\r \u00a0Cdc42.\t\r \u00a0Several\t\r \u00a0bacterial\t\r \u00a0effectors\t\r \u00a0are\t\r \u00a0known\t\r \u00a0to\t\r \u00a0be\t\r \u00a0polyubiquitylated\t\r \u00a0in\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0so\t\r \u00a0that\t\r \u00a0they\t\r \u00a0are\t\r \u00a0targeted\t\r \u00a0for\t\r \u00a0degradation,\t\r \u00a0and\t\r \u00a0some\t\r \u00a0also\t\r \u00a0mimic\t\r \u00a0various\t\r \u00a0components\t\r \u00a0of\t\r \u00a0the\t\r \u00a0ubiquitylation\t\r \u00a0machinery(474).\t\r \u00a0Within\t\r \u00a0the\t\r \u00a0past\t\r \u00a0few\t\r \u00a0years\t\r \u00a0great\t\r \u00a0progress\t\r \u00a0has\t\r \u00a0 \t\r \u00a0\t\r \u00a0 84\t\r \u00a0 been\t\r \u00a0made\t\r \u00a0towards\t\r \u00a0understanding\t\r \u00a0the\t\r \u00a0function\t\r \u00a0of\t\r \u00a0protein\t\r \u00a0monoubiquitylation,\t\r \u00a0and\t\r \u00a0it\t\r \u00a0is\t\r \u00a0now\t\r \u00a0widely\t\r \u00a0accepted\t\r \u00a0that\t\r \u00a0this\t\r \u00a0modification\t\r \u00a0can\t\r \u00a0serve\t\r \u00a0as\t\r \u00a0a\t\r \u00a0strong\t\r \u00a0endosomal\t\r \u00a0targeting\t\r \u00a0signal\t\r \u00a0(475-\u00ad\u2010477).\t\r \u00a0Previous\t\r \u00a0work\t\r \u00a0from\t\r \u00a0our\t\r \u00a0laboratory\t\r \u00a0and\t\r \u00a0others\t\r \u00a0has\t\r \u00a0demonstrated\t\r \u00a0that\t\r \u00a0SopB\t\r \u00a0remains\t\r \u00a0in\t\r \u00a0the\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0for\t\r \u00a0several\t\r \u00a0hours,\t\r \u00a0as\t\r \u00a0opposed\t\r \u00a0to\t\r \u00a0SopE,\t\r \u00a0which\t\r \u00a0is\t\r \u00a0rapidly\t\r \u00a0degraded\t\r \u00a0by\t\r \u00a0the\t\r \u00a0proteasome(196,\t\r \u00a0458).\t\r \u00a0Our\t\r \u00a0data\t\r \u00a0suggest\t\r \u00a0that\t\r \u00a0SopB\t\r \u00a0is\t\r \u00a0mostly\t\r \u00a0monoubiquitylated\t\r \u00a0and\t\r \u00a0that\t\r \u00a0there\t\r \u00a0are\t\r \u00a0at\t\r \u00a0least\t\r \u00a0two\t\r \u00a0sites\t\r \u00a0on\t\r \u00a0the\t\r \u00a0protein\t\r \u00a0where\t\r \u00a0this\t\r \u00a0occurs.\t\r \u00a0It\t\r \u00a0is\t\r \u00a0likely\t\r \u00a0that\t\r \u00a0there\t\r \u00a0is\t\r \u00a0at\t\r \u00a0least\t\r \u00a0one\t\r \u00a0more\t\r \u00a0ubiquitylation\t\r \u00a0site\t\r \u00a0between\t\r \u00a0residues\t\r \u00a0117\t\r \u00a0and\t\r \u00a0168,\t\r \u00a0since\t\r \u00a0SopB29-\u00ad\u2010168-\u00ad\u2010GFP\t\r \u00a0is\t\r \u00a0present\t\r \u00a0as\t\r \u00a0at\t\r \u00a0least\t\r \u00a0two\t\r \u00a0and\t\r \u00a0usually\t\r \u00a0three\t\r \u00a0or\t\r \u00a0four\t\r \u00a0bands\t\r \u00a0separated\t\r \u00a0by\t\r \u00a0approximately\t\r \u00a08\t\r \u00a0kDa,\t\r \u00a0while\t\r \u00a0GFP-\u00ad\u2010SopB29-\u00ad\u2010116\t\r \u00a0presented\t\r \u00a0as\t\r \u00a0a\t\r \u00a0monomer\t\r \u00a0on\t\r \u00a0SDS-\u00ad\u2010PAGE\t\r \u00a0(Figure\t\r \u00a02.6).\t\r \u00a0However,\t\r \u00a0these\t\r \u00a0ubiquitylation\t\r \u00a0events\t\r \u00a0could\t\r \u00a0be\t\r \u00a0forced\t\r \u00a0by\t\r \u00a0the\t\r \u00a0elimination\t\r \u00a0of\t\r \u00a0other\t\r \u00a0more\t\r \u00a0preferred\t\r \u00a0sites\t\r \u00a0in\t\r \u00a0the\t\r \u00a0N\t\r \u00a0and\t\r \u00a0C-\u00ad\u2010termini\t\r \u00a0as\t\r \u00a0is\t\r \u00a0frequently\t\r \u00a0seen\t\r \u00a0with\t\r \u00a0site-\u00ad\u2010directed\t\r \u00a0mutagenesis\t\r \u00a0studies(478)\t\r \u00a0since\t\r \u00a0full-\u00ad\u2010length\t\r \u00a0SopB\t\r \u00a0is\t\r \u00a0detected\t\r \u00a0primarily\t\r \u00a0with\t\r \u00a0zero\t\r \u00a0or\t\r \u00a0one\t\r \u00a0monoubiquitylations\t\r \u00a0(Figure\t\r \u00a02.8).\t\r \u00a0With\t\r \u00a0our\t\r \u00a0data\t\r \u00a0and\t\r \u00a0previous\t\r \u00a0reports\t\r \u00a0there\t\r \u00a0is\t\r \u00a0now\t\r \u00a0substantial\t\r \u00a0evidence\t\r \u00a0supporting\t\r \u00a0a\t\r \u00a0physical\t\r \u00a0interaction\t\r \u00a0of\t\r \u00a0SopB\t\r \u00a0with\t\r \u00a0the\t\r \u00a0N-\u00ad\u2010terminal\t\r \u00a0region\t\r \u00a0of\t\r \u00a0Cdc42.\t\r \u00a0Over-\u00ad\u2010expression\t\r \u00a0of\t\r \u00a0Cdc42\t\r \u00a0such\t\r \u00a0as\t\r \u00a0we\t\r \u00a0used\t\r \u00a0here\t\r \u00a0is\t\r \u00a0known\t\r \u00a0to\t\r \u00a0activate\t\r \u00a0the\t\r \u00a0protein,\t\r \u00a0and\t\r \u00a0indeed\t\r \u00a0we\t\r \u00a0observe\t\r \u00a0Cdc42\t\r \u00a0in\t\r \u00a0structures\t\r \u00a0near\t\r \u00a0the\t\r \u00a0membrane\t\r \u00a0that\t\r \u00a0resembled\t\r \u00a0ruffles.\t\r \u00a0When\t\r \u00a0SopB\t\r \u00a0is\t\r \u00a0co-\u00ad\u2010transfected\t\r \u00a0it\t\r \u00a0also\t\r \u00a0very\t\r \u00a0clearly\t\r \u00a0localizes\t\r \u00a0to\t\r \u00a0this\t\r \u00a0same\t\r \u00a0area,\t\r \u00a0in\t\r \u00a0contrast\t\r \u00a0to\t\r \u00a0a\t\r \u00a0previous\t\r \u00a0report\t\r \u00a0where\t\r \u00a0heterologously\t\r \u00a0expressed\t\r \u00a0SopB\t\r \u00a0localized\t\r \u00a0to\t\r \u00a0endosomes(200).\t\r \u00a0The\t\r \u00a0most\t\r \u00a0obvious\t\r \u00a0and\t\r \u00a0very\t\r \u00a0significant\t\r \u00a0difference\t\r \u00a0between\t\r \u00a0our\t\r \u00a0study\t\r \u00a0and\t\r \u00a0that\t\r \u00a0of\t\r \u00a0Dukes\t\r \u00a0et\t\r \u00a0al.\t\r \u00a0is\t\r \u00a0the\t\r \u00a0activation\t\r \u00a0state,\t\r \u00a0and\t\r \u00a0therefore\t\r \u00a0localization,\t\r \u00a0of\t\r \u00a0Cdc42.\t\r \u00a0In\t\r \u00a0separate\t\r \u00a0work\t\r \u00a0we\t\r \u00a0see\t\r \u00a0Cdc42\t\r \u00a0associating\t\r \u00a0with\t\r \u00a0maturing\t\r \u00a0phagosomes(479),\t\r \u00a0 \t\r \u00a0\t\r \u00a0 85\t\r \u00a0 suggesting\t\r \u00a0that\t\r \u00a0the\t\r \u00a0reported\t\r \u00a0endosomal\t\r \u00a0localization\t\r \u00a0of\t\r \u00a0SopB\t\r \u00a0occurs\t\r \u00a0after\t\r \u00a0the\t\r \u00a0initial\t\r \u00a0burst\t\r \u00a0of\t\r \u00a0Cdc42\t\r \u00a0activity\t\r \u00a0near\t\r \u00a0the\t\r \u00a0membrane\t\r \u00a0at\t\r \u00a0sites\t\r \u00a0of\t\r \u00a0invasion\/phagocytosis.\t\r \u00a0Subsequent\t\r \u00a0endosomal\t\r \u00a0targeting\t\r \u00a0of\t\r \u00a0monoubiquitylated\t\r \u00a0SopB\t\r \u00a0may\t\r \u00a0contribute\t\r \u00a0to\t\r \u00a0the\t\r \u00a0continued\t\r \u00a0role\t\r \u00a0of\t\r \u00a0SopB\t\r \u00a0in\t\r \u00a0SCV\t\r \u00a0trafficking\t\r \u00a0beyond\t\r \u00a0the\t\r \u00a0initial\t\r \u00a0invasion,\t\r \u00a0and\t\r \u00a0may\t\r \u00a0tie\t\r \u00a0in\t\r \u00a0with\t\r \u00a0the\t\r \u00a0potential\t\r \u00a0monoubiquitylation\t\r \u00a0described\t\r \u00a0here.\t\r \u00a0With\t\r \u00a0our\t\r \u00a0data\t\r \u00a0a\t\r \u00a0model\t\r \u00a0starts\t\r \u00a0to\t\r \u00a0coalesce,\t\r \u00a0whereby\t\r \u00a0the\t\r \u00a0SopB-\u00ad\u2010Cdc42\t\r \u00a0interaction\t\r \u00a0serves\t\r \u00a0to\t\r \u00a0localize\t\r \u00a0the\t\r \u00a0effector\t\r \u00a0to\t\r \u00a0sites\t\r \u00a0of\t\r \u00a0Cdc42\t\r \u00a0concentration\t\r \u00a0so\t\r \u00a0that\t\r \u00a0the\t\r \u00a0phosphoinositide\t\r \u00a0products\t\r \u00a0of\t\r \u00a0SopB\u2019s\t\r \u00a0enzymatic\t\r \u00a0activity\t\r \u00a0can\t\r \u00a0exert\t\r \u00a0their\t\r \u00a0effects.\t\r \u00a0The\t\r \u00a0specificity\t\r \u00a0SILAC\t\r \u00a0allows\t\r \u00a0for\t\r \u00a0the\t\r \u00a0identification\t\r \u00a0of\t\r \u00a0effector\t\r \u00a0targets\t\r \u00a0within\t\r \u00a0a\t\r \u00a0host\t\r \u00a0model,\t\r \u00a0and\t\r \u00a0should\t\r \u00a0now\t\r \u00a0enable\t\r \u00a0large-\u00ad\u2010scale\t\r \u00a0identification\t\r \u00a0of\t\r \u00a0host-\u00ad\u2010pathogen\t\r \u00a0interactions\t\r \u00a0for\t\r \u00a0a\t\r \u00a0wide\t\r \u00a0range\t\r \u00a0of\t\r \u00a0both\t\r \u00a0type\t\r \u00a0III\t\r \u00a0and\t\r \u00a0type\t\r \u00a0IV\t\r \u00a0effectors.\t\r \u00a0 \t\r \u00a0\t\r \u00a0 86\t\r \u00a0 3\t\r \u00a0An\t\r \u00a0integrated\t\r \u00a0global\t\r \u00a0strategy\t\r \u00a0for\t\r \u00a0cell\t\r \u00a0lysis,\t\r \u00a0fractionation,\t\r \u00a0 enrichment\t\r \u00a0and\t\r \u00a0mass\t\r \u00a0spectrometric\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0 phosphorylated\t\r \u00a0peptides\t\r \u00a0 3.1\t\r \u00a0Introduction\t\r \u00a0Reversible\t\r \u00a0protein\t\r \u00a0phosphorylation\t\r \u00a0on\t\r \u00a0serine,\t\r \u00a0threonine,\t\r \u00a0and\t\r \u00a0tyrosine\t\r \u00a0residues\t\r \u00a0is\t\r \u00a0believed\t\r \u00a0to\t\r \u00a0be\t\r \u00a0among\t\r \u00a0the\t\r \u00a0most\t\r \u00a0widespread\t\r \u00a0post-\u00ad\u2010translational\t\r \u00a0modifications\t\r \u00a0of\t\r \u00a0proteins\t\r \u00a0and\t\r \u00a0has\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0regulate\t\r \u00a0a\t\r \u00a0vast\t\r \u00a0array\t\r \u00a0of\t\r \u00a0cellular\t\r \u00a0processes(380,\t\r \u00a0480).\t\r \u00a0Recently,\t\r \u00a0studying\t\r \u00a0protein\t\r \u00a0phosphorylation\t\r \u00a0has\t\r \u00a0developed\t\r \u00a0from\t\r \u00a0almost\t\r \u00a0exclusively\t\r \u00a0a\t\r \u00a0reductionist\t\r \u00a0approach,\t\r \u00a0to\t\r \u00a0global\t\r \u00a0analyses\t\r \u00a0capable\t\r \u00a0of\t\r \u00a0tracking\t\r \u00a0thousands\t\r \u00a0of\t\r \u00a0phosphorylation\t\r \u00a0events\t\r \u00a0simultaneously(297,\t\r \u00a0390,\t\r \u00a0418).\t\r \u00a0Originally,\t\r \u00a02DE\t\r \u00a0utilizing\t\r \u00a032P-\u00ad\u2010labeled\t\r \u00a0proteins\t\r \u00a0or\t\r \u00a0colorimetric\t\r \u00a0and\t\r \u00a0fluorimetric\t\r \u00a0phosphate\t\r \u00a0specific\t\r \u00a0imaging\t\r \u00a0to\t\r \u00a0detect\t\r \u00a0and\t\r \u00a0visualize\t\r \u00a0phosphoproteins\t\r \u00a0was\t\r \u00a0the\t\r \u00a0method\t\r \u00a0of\t\r \u00a0choice\t\r \u00a0for\t\r \u00a0phosphorylation\t\r \u00a0analysis\t\r \u00a0on\t\r \u00a0a\t\r \u00a0proteome\t\r \u00a0scale\t\r \u00a0(i.e.,\t\r \u00a0phosphoproteomics)(481,\t\r \u00a0482).\t\r \u00a0Despite\t\r \u00a0the\t\r \u00a0high\t\r \u00a0sensitivy\t\r \u00a0of\t\r \u00a0these\t\r \u00a0imaging\t\r \u00a0techniques,\t\r \u00a0relatively\t\r \u00a0low\t\r \u00a0throughput\t\r \u00a0and\t\r \u00a0sensitivity\t\r \u00a0in\t\r \u00a0spot\t\r \u00a0identification\t\r \u00a0has\t\r \u00a0led\t\r \u00a0to\t\r \u00a0the\t\r \u00a0replacement\t\r \u00a0of\t\r \u00a0several\t\r \u00a02DE\t\r \u00a0approaches\t\r \u00a0by\t\r \u00a0LC-\u00ad\u2010MSn.\t\r \u00a0However,\t\r \u00a0even\t\r \u00a0using\t\r \u00a0these\t\r \u00a0new\t\r \u00a0approaches,\t\r \u00a0its\t\r \u00a0reversible\t\r \u00a0nature,\t\r \u00a0the\t\r \u00a0low\t\r \u00a0stoichiometry\t\r \u00a0of\t\r \u00a0protein\t\r \u00a0phosphorylation\t\r \u00a0events,\t\r \u00a0and\t\r \u00a0the\t\r \u00a0lability\t\r \u00a0of\t\r \u00a0the\t\r \u00a0phosphate\t\r \u00a0are\t\r \u00a0still\t\r \u00a0considered\t\r \u00a0significant\t\r \u00a0challenges\t\r \u00a0in\t\r \u00a0phosphoproteomics.\t\r \u00a0Due\t\r \u00a0to\t\r \u00a0the\t\r \u00a0reversible\t\r \u00a0nature\t\r \u00a0of\t\r \u00a0protein\t\r \u00a0phosphorylation,\t\r \u00a0phosphatases\t\r \u00a0liberated\t\r \u00a0upon\t\r \u00a0cell\t\r \u00a0lysis\t\r \u00a0can\t\r \u00a0quickly\t\r \u00a0and\t\r \u00a0substantially\t\r \u00a0reduce\t\r \u00a0the\t\r \u00a0signal\t\r \u00a0from\t\r \u00a0a\t\r \u00a0given\t\r \u00a0stimulus.\t\r \u00a0By\t\r \u00a0far\t\r \u00a0the\t\r \u00a0most\t\r \u00a0common\t\r \u00a0means\t\r \u00a0of\t\r \u00a0inhibiting\t\r \u00a0phosphatases\t\r \u00a0is\t\r \u00a0through\t\r \u00a0the\t\r \u00a0 \t\r \u00a0\t\r \u00a0 87\t\r \u00a0 use\t\r \u00a0of\t\r \u00a0commercially\t\r \u00a0available\t\r \u00a0inhibitors.\t\r \u00a0Vanadium\t\r \u00a0oxides\t\r \u00a0such\t\r \u00a0as\t\r \u00a0pervanadate\t\r \u00a0and\t\r \u00a0orthovanadate\t\r \u00a0are\t\r \u00a0typically\t\r \u00a0used\t\r \u00a0to\t\r \u00a0inhibit\t\r \u00a0PTPs.\t\r \u00a0Of\t\r \u00a0the\t\r \u00a0twenty\t\r \u00a0known\t\r \u00a0families\t\r \u00a0of\t\r \u00a0serine\/threonine\t\r \u00a0phosphatases,\t\r \u00a0the\t\r \u00a0most\t\r \u00a0common\t\r \u00a0are\t\r \u00a0PP1,\t\r \u00a0PP2A\t\r \u00a0and\t\r \u00a0PP2B,\t\r \u00a0which\t\r \u00a0are\t\r \u00a0targeted\t\r \u00a0by\t\r \u00a0common\t\r \u00a0inhibitors\t\r \u00a0such\t\r \u00a0as\t\r \u00a0calyculin\t\r \u00a0A\t\r \u00a0(PP1\t\r \u00a0and\t\r \u00a0PP2A)\t\r \u00a0and\t\r \u00a0deltamethrin\t\r \u00a0(PP2B).\t\r \u00a0However,\t\r \u00a0while\t\r \u00a0treating\t\r \u00a0live\t\r \u00a0Hepa1-\u00ad\u20106\t\r \u00a0liver\t\r \u00a0cells\t\r \u00a0with\t\r \u00a0pervanadate,\t\r \u00a0calyculin\t\r \u00a0A,\t\r \u00a0and\t\r \u00a0deltamethrin,\t\r \u00a0Pan\t\r \u00a0et\t\r \u00a0al.\t\r \u00a0compared\t\r \u00a0the\t\r \u00a0abundance\t\r \u00a0of\t\r \u00a0individual\t\r \u00a0phosphorylated\t\r \u00a0peptides\t\r \u00a0to\t\r \u00a0an\t\r \u00a0untreated\t\r \u00a0condition\t\r \u00a0and\t\r \u00a0observed\t\r \u00a0that\t\r \u00a0only\t\r \u00a027%\t\r \u00a0increased\t\r \u00a0more\t\r \u00a0than\t\r \u00a0two-\u00ad\u2010fold(387).\t\r \u00a0Similarly,\t\r \u00a0in\t\r \u00a0an\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0the\t\r \u00a0stem\t\r \u00a0cell\t\r \u00a0plasma\t\r \u00a0membrane\t\r \u00a0phosphoproteome,\t\r \u00a0Thingholm\t\r \u00a0et\t\r \u00a0al.\t\r \u00a0found\t\r \u00a0that\t\r \u00a0pretreating\t\r \u00a0cells\t\r \u00a0with\t\r \u00a0calyculin\t\r \u00a0A,\t\r \u00a0sodium\t\r \u00a0pervanadate,\t\r \u00a0or\t\r \u00a0two\t\r \u00a0commercially\t\r \u00a0available\t\r \u00a0phosphatase\t\r \u00a0inhibitor\t\r \u00a0cocktails\t\r \u00a0resulted\t\r \u00a0in\t\r \u00a0only\t\r \u00a0a\t\r \u00a010-\u00ad\u201040%\t\r \u00a0increase\t\r \u00a0in\t\r \u00a0the\t\r \u00a0number\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0identified(388).\t\r \u00a0In\t\r \u00a0both\t\r \u00a0cases,\t\r \u00a0a\t\r \u00a0majority\t\r \u00a0of\t\r \u00a0pY\t\r \u00a0sites\t\r \u00a0were\t\r \u00a0preserved,\t\r \u00a0while\t\r \u00a0pS\t\r \u00a0and\t\r \u00a0pT\t\r \u00a0sites\t\r \u00a0were\t\r \u00a0less\t\r \u00a0effectively\t\r \u00a0protected\t\r \u00a0by\t\r \u00a0the\t\r \u00a0inhibitors.\t\r \u00a0Thus,\t\r \u00a0while\t\r \u00a0inhibitor-\u00ad\u2010based\t\r \u00a0methods\t\r \u00a0are\t\r \u00a0somewhat\t\r \u00a0effective,\t\r \u00a0especially\t\r \u00a0when\t\r \u00a0considering\t\r \u00a0tyrosine\t\r \u00a0phosphorylation,\t\r \u00a0truly\t\r \u00a0global\t\r \u00a0analyses\t\r \u00a0require\t\r \u00a0additional\t\r \u00a0techniques.\t\r \u00a0Due\t\r \u00a0to\t\r \u00a0the\t\r \u00a0tight\t\r \u00a0spatial\t\r \u00a0and\t\r \u00a0temporal\t\r \u00a0control\t\r \u00a0observed\t\r \u00a0in\t\r \u00a0signalling\t\r \u00a0pathways,\t\r \u00a0protein\t\r \u00a0phosphorylation\t\r \u00a0events\t\r \u00a0typically\t\r \u00a0occur\t\r \u00a0at\t\r \u00a0very\t\r \u00a0low\t\r \u00a0stoichiometry(480,\t\r \u00a0483).\t\r \u00a0Thus\t\r \u00a0fractionation\t\r \u00a0and\t\r \u00a0enrichment\t\r \u00a0of\t\r \u00a0phosphorylated\t\r \u00a0peptides\t\r \u00a0and\t\r \u00a0proteins\t\r \u00a0prior\t\r \u00a0to\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0analysis\t\r \u00a0is\t\r \u00a0indispensable\t\r \u00a0for\t\r \u00a0large-\u00ad\u2010scale\t\r \u00a0phosphoproteomics.\t\r \u00a0In\t\r \u00a0addition\t\r \u00a0to\t\r \u00a0subcellular\t\r \u00a0fractionation\t\r \u00a0to\t\r \u00a0purify\t\r \u00a0a\t\r \u00a0given\t\r \u00a0organelle\t\r \u00a0and\t\r \u00a0calcium\t\r \u00a0phosphate\t\r \u00a0precipitation\t\r \u00a0or\t\r \u00a0barium\t\r \u00a0phosphate\t\r \u00a0precipitation\t\r \u00a0through\t\r \u00a0a\t\r \u00a0pH\t\r \u00a0range,\t\r \u00a0several\t\r \u00a0chromatographic\t\r \u00a0approaches\t\r \u00a0have\t\r \u00a0been\t\r \u00a0reported\t\r \u00a0to\t\r \u00a0 \t\r \u00a0\t\r \u00a0 88\t\r \u00a0 enrich\t\r \u00a0phosphopeptides\t\r \u00a0based\t\r \u00a0on\t\r \u00a0either\t\r \u00a0their\t\r \u00a0negative\t\r \u00a0charge\t\r \u00a0or\t\r \u00a0polarity(297,\t\r \u00a0398,\t\r \u00a0399).\t\r \u00a0These\t\r \u00a0include\t\r \u00a0SCX\t\r \u00a0and\t\r \u00a0SAX\t\r \u00a0chromatography,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0HILIC\t\r \u00a0and\t\r \u00a0ERLIC(359,\t\r \u00a0389-\u00ad\u2010391,\t\r \u00a0395-\u00ad\u2010397).\t\r \u00a0To\t\r \u00a0a\t\r \u00a0lesser\t\r \u00a0extent,\t\r \u00a0IEF\t\r \u00a0has\t\r \u00a0also\t\r \u00a0been\t\r \u00a0used\t\r \u00a0to\t\r \u00a0enrich\t\r \u00a0for\t\r \u00a0phosphopeptides\t\r \u00a0in\t\r \u00a0the\t\r \u00a0low\t\r \u00a0pH\t\r \u00a0range(400-\u00ad\u2010402,\t\r \u00a0484).\t\r \u00a0SCX\t\r \u00a0is\t\r \u00a0by\t\r \u00a0far\t\r \u00a0the\t\r \u00a0most\t\r \u00a0commonly\t\r \u00a0used\t\r \u00a0technique\t\r \u00a0for\t\r \u00a0phosphopeptide\t\r \u00a0pre-\u00ad\u2010fractionation,\t\r \u00a0as\t\r \u00a0the\t\r \u00a0strong\t\r \u00a0negative\t\r \u00a0charge\t\r \u00a0on\t\r \u00a0phosphate\t\r \u00a0causes\t\r \u00a0phosphopeptides\t\r \u00a0to\t\r \u00a0elute\t\r \u00a0earlier\t\r \u00a0than\t\r \u00a0the\t\r \u00a0majority\t\r \u00a0of\t\r \u00a0nonphosphorylated\t\r \u00a0peptides.\t\r \u00a0However,\t\r \u00a0with\t\r \u00a0several\t\r \u00a0chromatographic\t\r \u00a0matrices\t\r \u00a0(i.e.\t\r \u00a0SAX\t\r \u00a0and\t\r \u00a0HILIC\t\r \u00a0resins\t\r \u00a0where\t\r \u00a0phosphopeptides\t\r \u00a0bind\t\r \u00a0very\t\r \u00a0strongly\t\r \u00a0and\t\r \u00a0are\t\r \u00a0difficult\t\r \u00a0to\t\r \u00a0elute(482))\t\r \u00a0sample\t\r \u00a0is\t\r \u00a0lost\t\r \u00a0either\t\r \u00a0in\t\r \u00a0the\t\r \u00a0flow\t\r \u00a0through\t\r \u00a0or\t\r \u00a0by\t\r \u00a0irreversibly\t\r \u00a0binding\t\r \u00a0the\t\r \u00a0resin.\t\r \u00a0Precipitation\t\r \u00a0methods\t\r \u00a0can\t\r \u00a0also\t\r \u00a0result\t\r \u00a0in\t\r \u00a0similar\t\r \u00a0sample\t\r \u00a0losses,\t\r \u00a0thus\t\r \u00a0fractionation\t\r \u00a0procedures\t\r \u00a0such\t\r \u00a0as\t\r \u00a0in-\u00ad\u2010solution\t\r \u00a0IEF\t\r \u00a0offer\t\r \u00a0the\t\r \u00a0advantage\t\r \u00a0of\t\r \u00a0virtually\t\r \u00a0complete\t\r \u00a0sample\t\r \u00a0recovery.\t\r \u00a0Following\t\r \u00a0a\t\r \u00a0pre-\u00ad\u2010fractionation\t\r \u00a0step,\t\r \u00a0most\t\r \u00a0studies\t\r \u00a0employ\t\r \u00a0one\t\r \u00a0of\t\r \u00a0a\t\r \u00a0few\t\r \u00a0approaches\t\r \u00a0offering\t\r \u00a0much\t\r \u00a0higher\t\r \u00a0selectivity\t\r \u00a0for\t\r \u00a0phosphopeptides.\t\r \u00a0While\t\r \u00a0immunoprecipitation\t\r \u00a0of\t\r \u00a0tyrosine\t\r \u00a0phosphorylated\t\r \u00a0species\t\r \u00a0has\t\r \u00a0proven\t\r \u00a0effective,\t\r \u00a0poor\t\r \u00a0specificity\t\r \u00a0has\t\r \u00a0generally\t\r \u00a0been\t\r \u00a0observed\t\r \u00a0for\t\r \u00a0pS\t\r \u00a0and\t\r \u00a0pT\t\r \u00a0antibodies(331,\t\r \u00a0485-\u00ad\u2010487).\t\r \u00a0IMAC\t\r \u00a0is\t\r \u00a0based\t\r \u00a0on\t\r \u00a0the\t\r \u00a0high\t\r \u00a0affinity\t\r \u00a0of\t\r \u00a0phosphate\t\r \u00a0for\t\r \u00a0metal\t\r \u00a0ions\t\r \u00a0such\t\r \u00a0as\t\r \u00a0Fe3+,\t\r \u00a0Zn2+,\t\r \u00a0and\t\r \u00a0Ga3+\t\r \u00a0and\t\r \u00a0has\t\r \u00a0been\t\r \u00a0extensively\t\r \u00a0used,\t\r \u00a0often\t\r \u00a0incorporating\t\r \u00a0methyl\t\r \u00a0esterification\t\r \u00a0of\t\r \u00a0carboxylates\t\r \u00a0which\t\r \u00a0otherwise\t\r \u00a0also\t\r \u00a0bind\t\r \u00a0strongly\t\r \u00a0to\t\r \u00a0the\t\r \u00a0metal\t\r \u00a0ions(375,\t\r \u00a0406,\t\r \u00a0407,\t\r \u00a0410).\t\r \u00a0Alternatively,\t\r \u00a0MOC\t\r \u00a0offers\t\r \u00a0very\t\r \u00a0selective\t\r \u00a0enrichment\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0and\t\r \u00a0is\t\r \u00a0based\t\r \u00a0on\t\r \u00a0the\t\r \u00a0affinity\t\r \u00a0of\t\r \u00a0acidified\t\r \u00a0phosphoric\t\r \u00a0acid\t\r \u00a0for\t\r \u00a0metal\t\r \u00a0oxides\t\r \u00a0such\t\r \u00a0as\t\r \u00a0TiO2\t\r \u00a0and\t\r \u00a0ZrO2(411).\t\r \u00a0Without\t\r \u00a0requiring\t\r \u00a0chemical\t\r \u00a0derivitization,\t\r \u00a0MOC\t\r \u00a0most\t\r \u00a0often\t\r \u00a0utilizes\t\r \u00a0DHB\t\r \u00a0to\t\r \u00a0selectively\t\r \u00a0compete\t\r \u00a0off\t\r \u00a0carboxylate\t\r \u00a0containing\t\r \u00a0peptides\t\r \u00a0from\t\r \u00a0the\t\r \u00a0TiO2\t\r \u00a0or\t\r \u00a0ZrO2\t\r \u00a0 \t\r \u00a0\t\r \u00a0 89\t\r \u00a0 matrix(376).\t\r \u00a0However,\t\r \u00a0as\t\r \u00a0even\t\r \u00a0small\t\r \u00a0amouts\t\r \u00a0of\t\r \u00a0DHB\t\r \u00a0can\t\r \u00a0accumulate\t\r \u00a0and\t\r \u00a0damage\t\r \u00a0electrospray\t\r \u00a0systems,\t\r \u00a0aliphatic\t\r \u00a0hydroxyl\t\r \u00a0acids\t\r \u00a0such\t\r \u00a0as\t\r \u00a0lactic\t\r \u00a0acid\t\r \u00a0or\t\r \u00a0\u03b2-\u00ad\u2010hydroxypropanoic\t\r \u00a0acid\t\r \u00a0can\t\r \u00a0also\t\r \u00a0be\t\r \u00a0used(412).\t\r \u00a0CID\t\r \u00a0is\t\r \u00a0often\t\r \u00a0considered\t\r \u00a0suboptimal\t\r \u00a0for\t\r \u00a0phosphoproteomics,\t\r \u00a0as\t\r \u00a0neutral\t\r \u00a0loss\t\r \u00a0of\t\r \u00a0phosphoric\t\r \u00a0acid\t\r \u00a0(H3PO4\t\r \u00a0from\t\r \u00a0pS\t\r \u00a0and\t\r \u00a0pT)\t\r \u00a0can\t\r \u00a0occur\t\r \u00a0before\t\r \u00a0backbone\t\r \u00a0cleavage,\t\r \u00a0resulting\t\r \u00a0in\t\r \u00a0insufficient\t\r \u00a0backbone\t\r \u00a0fragmentation\t\r \u00a0for\t\r \u00a0effective\t\r \u00a0identification\t\r \u00a0of\t\r \u00a0the\t\r \u00a0peptide(419,\t\r \u00a0488).\t\r \u00a0Methods\t\r \u00a0such\t\r \u00a0as\t\r \u00a0MS3\t\r \u00a0and\t\r \u00a0MultiStage\t\r \u00a0Activation\t\r \u00a0impose\t\r \u00a0additional\t\r \u00a0activation\t\r \u00a0events\t\r \u00a0on\t\r \u00a0preselected\t\r \u00a0neutral\t\r \u00a0loss\t\r \u00a0peaks(389,\t\r \u00a0420).\t\r \u00a0While\t\r \u00a0the\t\r \u00a0majority\t\r \u00a0of\t\r \u00a0phosphoproteomics\t\r \u00a0studies\t\r \u00a0use\t\r \u00a0these\t\r \u00a0techniques\t\r \u00a0when\t\r \u00a0possible,\t\r \u00a0the\t\r \u00a0advantages\t\r \u00a0are\t\r \u00a0somewhat\t\r \u00a0controversial.\t\r \u00a0Villen\t\r \u00a0et\t\r \u00a0al.\t\r \u00a0have\t\r \u00a0shown\t\r \u00a0that,\t\r \u00a0when\t\r \u00a0compared\t\r \u00a0to\t\r \u00a0MS2,\t\r \u00a0MS3\t\r \u00a0and\t\r \u00a0pseudo-\u00ad\u2010MS3\t\r \u00a0schemes\t\r \u00a0resulted\t\r \u00a0in\t\r \u00a0an\t\r \u00a0overall\t\r \u00a0decrease\t\r \u00a0in\t\r \u00a0the\t\r \u00a0number\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0identified\t\r \u00a0and\t\r \u00a0offered\t\r \u00a0only\t\r \u00a0a\t\r \u00a0very\t\r \u00a0minor\t\r \u00a0advantage\t\r \u00a0in\t\r \u00a0phospho\t\r \u00a0site\t\r \u00a0localization(422).\t\r \u00a0More\t\r \u00a0recently\t\r \u00a0however,\t\r \u00a0Ulintz\t\r \u00a0et\t\r \u00a0al.\t\r \u00a0observed\t\r \u00a0that\t\r \u00a0MultiStage\t\r \u00a0Activation\t\r \u00a0did\t\r \u00a0increase\t\r \u00a0the\t\r \u00a0number\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0identified\t\r \u00a0when\t\r \u00a0compared\t\r \u00a0to\t\r \u00a0MS2\t\r \u00a0and\t\r \u00a0MS3\t\r \u00a0based\t\r \u00a0methodologies(423).\t\r \u00a0There\t\r \u00a0have\t\r \u00a0been\t\r \u00a0several\t\r \u00a0significant\t\r \u00a0technical\t\r \u00a0advances\t\r \u00a0in\t\r \u00a0phosphoproteomics\t\r \u00a0in\t\r \u00a0the\t\r \u00a0past\t\r \u00a0few\t\r \u00a0years(489);\t\r \u00a0nonetheless,\t\r \u00a0we\t\r \u00a0believe\t\r \u00a0there\t\r \u00a0are\t\r \u00a0still\t\r \u00a0significant\t\r \u00a0challenges\t\r \u00a0and\t\r \u00a0room\t\r \u00a0for\t\r \u00a0improvement.\t\r \u00a0To\t\r \u00a0this\t\r \u00a0end,\t\r \u00a0we\t\r \u00a0have\t\r \u00a0undertaken\t\r \u00a0a\t\r \u00a0comprehensive\t\r \u00a0re-\u00ad\u2010evaluation\t\r \u00a0of\t\r \u00a0all\t\r \u00a0the\t\r \u00a0significant\t\r \u00a0steps\t\r \u00a0involved\t\r \u00a0in\t\r \u00a0a\t\r \u00a0phosphoproteomics\t\r \u00a0experiment.\t\r \u00a0We\t\r \u00a0present\t\r \u00a0here\t\r \u00a0a\t\r \u00a0modified\t\r \u00a0strategy\t\r \u00a0with\t\r \u00a0significant\t\r \u00a0improvements\t\r \u00a0at\t\r \u00a0each\t\r \u00a0step,\t\r \u00a0beginning\t\r \u00a0with\t\r \u00a0improved\t\r \u00a0inhibition\t\r \u00a0of\t\r \u00a0phosphatases\t\r \u00a0during\t\r \u00a0cell\t\r \u00a0lysis\t\r \u00a0and\t\r \u00a0ending\t\r \u00a0with\t\r \u00a0an\t\r \u00a0evaluation\t\r \u00a0of\t\r \u00a0different\t\r \u00a0fragmentation\t\r \u00a0mechanisms\t\r \u00a0for\t\r \u00a0the\t\r \u00a0purpose\t\r \u00a0of\t\r \u00a0identifying\t\r \u00a0phosphopeptides.\t\r \u00a0 \t\r \u00a0\t\r \u00a0 90\t\r \u00a0 \t\r \u00a0 3.2\t\r \u00a0Experimental\t\r \u00a0procedures\t\r \u00a0 3.2.1\t\r \u00a0Cell\t\r \u00a0culture\t\r \u00a0and\t\r \u00a0lysis\t\r \u00a0HeLa\t\r \u00a0cells\t\r \u00a0were\t\r \u00a0maintained\t\r \u00a0in\t\r \u00a0DMEM\t\r \u00a0containing\t\r \u00a04500\t\r \u00a0mg\/L\t\r \u00a0glucose\t\r \u00a0and\t\r \u00a04\t\r \u00a0mM\t\r \u00a01-\u00ad\u2010glutamine\t\r \u00a0(Thermo\t\r \u00a0Fisher\t\r \u00a0Scientific),\t\r \u00a0supplemented\t\r \u00a0with\t\r \u00a010%\t\r \u00a0v\/v\t\r \u00a0qualified\t\r \u00a0FBS\t\r \u00a0(Invitrogen),\t\r \u00a0an\t\r \u00a0additional\t\r \u00a02\t\r \u00a0mM\t\r \u00a01-\u00ad\u2010glutamine\t\r \u00a0(Thermo\t\r \u00a0Fisher\t\r \u00a0Scientific)\t\r \u00a0and\t\r \u00a00.1\t\r \u00a0units\/L\t\r \u00a0penicillin\t\r \u00a0and\t\r \u00a0streptomycin\t\r \u00a0(Thermo\t\r \u00a0Fisher\t\r \u00a0Scientific).\t\r \u00a0For\t\r \u00a0harvesting\t\r \u00a0and\t\r \u00a0lysis,\t\r \u00a0cells\t\r \u00a0were\t\r \u00a0placed\t\r \u00a0on\t\r \u00a0ice,\t\r \u00a0washed\t\r \u00a03x\t\r \u00a0with\t\r \u00a0cold\t\r \u00a0PBS\t\r \u00a0and\t\r \u00a0harvested\t\r \u00a0with\t\r \u00a0a\t\r \u00a0scraper.\t\r \u00a0Cells\t\r \u00a0were\t\r \u00a0pelleted\t\r \u00a0at\t\r \u00a0600\t\r \u00a0relative\t\r \u00a0centrifugal\t\r \u00a0force\t\r \u00a0(rcf)\t\r \u00a0for\t\r \u00a05\t\r \u00a0min\t\r \u00a0at\t\r \u00a04\u00b0C.\t\r \u00a0For\t\r \u00a0all\t\r \u00a0experiments\t\r \u00a0described,\t\r \u00a0excluding\t\r \u00a0the\t\r \u00a0phosphatase\t\r \u00a0activity\t\r \u00a0assay\t\r \u00a0and\t\r \u00a0experiments\t\r \u00a0analysing\t\r \u00a0the\t\r \u00a0inhibition\t\r \u00a0of\t\r \u00a0phosphopeptide\t\r \u00a0enrichment\t\r \u00a0by\t\r \u00a0phosphatase\t\r \u00a0inhibitors,\t\r \u00a0the\t\r \u00a0cell\t\r \u00a0pellet\t\r \u00a0was\t\r \u00a0resuspended\t\r \u00a0in\t\r \u00a0200\t\r \u00a0\u03bcL\t\r \u00a01%\t\r \u00a0w\/v\t\r \u00a0Na\t\r \u00a0deoxycholate,\t\r \u00a050\t\r \u00a0mM\t\r \u00a0NH4HCO3\t\r \u00a0per\t\r \u00a010\t\r \u00a0cm\t\r \u00a0plate.\t\r \u00a0The\t\r \u00a0lysate\t\r \u00a0was\t\r \u00a0then\t\r \u00a0immediately\t\r \u00a0placed\t\r \u00a0in\t\r \u00a0a\t\r \u00a0heating\t\r \u00a0block\t\r \u00a0at\t\r \u00a099\u00b0C\t\r \u00a0for\t\r \u00a05\t\r \u00a0min.\t\r \u00a0Samples\t\r \u00a0were\t\r \u00a0removed\t\r \u00a0and\t\r \u00a0cooled\t\r \u00a0to\t\r \u00a0room\t\r \u00a0temperature,\t\r \u00a0MgCl2\t\r \u00a0was\t\r \u00a0added\t\r \u00a0to\t\r \u00a0a\t\r \u00a0final\t\r \u00a0concentration\t\r \u00a0of\t\r \u00a01.5\t\r \u00a0mM\t\r \u00a0and\t\r \u00a02.5x10-\u00ad\u20103\t\r \u00a0units\/\u03bcL\t\r \u00a0benzonase\t\r \u00a0(Novagen)\t\r \u00a0were\t\r \u00a0added\t\r \u00a0to\t\r \u00a0cleave\t\r \u00a0DNA\t\r \u00a0and\t\r \u00a0decrease\t\r \u00a0viscosity.\t\r \u00a0\t\r \u00a0 3.2.2\t\r \u00a0Phosphate\t\r \u00a0assay\t\r \u00a0For\t\r \u00a0each\t\r \u00a0condition,\t\r \u00a0one\t\r \u00a010\t\r \u00a0cm\t\r \u00a0plate\t\r \u00a0of\t\r \u00a0HeLa\t\r \u00a0cells\t\r \u00a0was\t\r \u00a0washed\t\r \u00a03x\t\r \u00a0with\t\r \u00a0cold\t\r \u00a020\t\r \u00a0mM\t\r \u00a0tris,\t\r \u00a0150\t\r \u00a0mM\t\r \u00a0NaCl,\t\r \u00a0pH\t\r \u00a07.5,\t\r \u00a0harvested\t\r \u00a0with\t\r \u00a0a\t\r \u00a0scraper,\t\r \u00a0and\t\r \u00a0pelleted\t\r \u00a0at\t\r \u00a0600\t\r \u00a0rcf\t\r \u00a0for\t\r \u00a05\t\r \u00a0min\t\r \u00a0at\t\r \u00a04\u00b0C.\t\r \u00a0Equally\t\r \u00a0sized\t\r \u00a0cell\t\r \u00a0pellets\t\r \u00a0were\t\r \u00a0subjected\t\r \u00a0to\t\r \u00a0each\t\r \u00a0of\t\r \u00a0the\t\r \u00a0following\t\r \u00a0lysis\t\r \u00a0conditions.\t\r \u00a0Cell\t\r \u00a0pellets\t\r \u00a0subjected\t\r \u00a0to\t\r \u00a0the\t\r \u00a0\u2018No\t\r \u00a0Inhibitors\u2019\t\r \u00a0conditions\t\r \u00a0were\t\r \u00a0resuspended\t\r \u00a0and\t\r \u00a0lysed\t\r \u00a0in\t\r \u00a01%\t\r \u00a0v\/v\t\r \u00a0Nonidet\t\r \u00a0P40,\t\r \u00a020\t\r \u00a0mM\t\r \u00a0tris,\t\r \u00a0150\t\r \u00a0mM\t\r \u00a0NaCl,\t\r \u00a0and\t\r \u00a0protease\t\r \u00a0inhibitor\t\r \u00a0 \t\r \u00a0\t\r \u00a0 91\t\r \u00a0 cocktail,\t\r \u00a0pH\t\r \u00a07.5.\t\r \u00a0Cell\t\r \u00a0pellets\t\r \u00a0subjected\t\r \u00a0to\t\r \u00a0the\t\r \u00a0\u2018Inhibitors\u2019\t\r \u00a0conditions\t\r \u00a0were\t\r \u00a0resuspended\t\r \u00a0and\t\r \u00a0lysed\t\r \u00a0in\t\r \u00a01%\t\r \u00a0Nonidet\t\r \u00a0P40,\t\r \u00a020\t\r \u00a0mM\t\r \u00a0tris,\t\r \u00a0150\t\r \u00a0mM\t\r \u00a0NaCl,\t\r \u00a0protease\t\r \u00a0inhibitor\t\r \u00a0cocktail,\t\r \u00a0pH\t\r \u00a07.5\t\r \u00a0with\t\r \u00a0the\t\r \u00a0addition\t\r \u00a0of\t\r \u00a01\t\r \u00a0mM\t\r \u00a0Na3VO4,\t\r \u00a010\t\r \u00a0mM\t\r \u00a0NaF,\t\r \u00a00.5\t\r \u00a0mM\t\r \u00a0pervanadate,\t\r \u00a010\t\r \u00a0\u03bcM\t\r \u00a0deltamethrin,\t\r \u00a0100\t\r \u00a0nM\t\r \u00a0calyculin\t\r \u00a0A.\t\r \u00a0Cell\t\r \u00a0pellets\t\r \u00a0subjected\t\r \u00a0to\t\r \u00a0\u2018Deoxycholate+Inhibitors\u2019\t\r \u00a0conditions\t\r \u00a0were\t\r \u00a0resuspended\t\r \u00a0in\t\r \u00a020\t\r \u00a0mM\t\r \u00a0tris,\t\r \u00a0150\t\r \u00a0mM\t\r \u00a0NaCl,\t\r \u00a0protease\t\r \u00a0inihibitor\t\r \u00a0cocktail,\t\r \u00a0pH\t\r \u00a07.5\t\r \u00a0and\t\r \u00a0identical\t\r \u00a0phosphatase\t\r \u00a0inhibitors\t\r \u00a0and\t\r \u00a0concentrations\t\r \u00a0listed\t\r \u00a0above.\t\r \u00a0Cell\t\r \u00a0pellets\t\r \u00a0subjected\t\r \u00a0to\t\r \u00a0the\t\r \u00a0\u2018Deoxycholate\u2019\t\r \u00a0conditions\t\r \u00a0were\t\r \u00a0resuspended\t\r \u00a0in\t\r \u00a020mM\t\r \u00a0Tris,\t\r \u00a0150\t\r \u00a0mM\t\r \u00a0NaCl,\t\r \u00a0protease\t\r \u00a0inihibitor\t\r \u00a0cocktail,\t\r \u00a0pH\t\r \u00a07.5.\t\r \u00a0\t\r \u00a0Cells\t\r \u00a0pellets\t\r \u00a0subjected\t\r \u00a0to\t\r \u00a0the\t\r \u00a0\u2018Deoxycholate+Inhibitors\u2019\t\r \u00a0and\t\r \u00a0\u2018Deoxycholate\u2019\t\r \u00a0conditions\t\r \u00a0were\t\r \u00a0mechanically\t\r \u00a0lysed\t\r \u00a0by\t\r \u00a05-\u00ad\u20106\t\r \u00a0passages\t\r \u00a0through\t\r \u00a0a\t\r \u00a022G\t\r \u00a0needle.\t\r \u00a0Nuclei\t\r \u00a0were\t\r \u00a0pelleted\t\r \u00a0at\t\r \u00a0600\t\r \u00a0rcf\t\r \u00a0for\t\r \u00a05\t\r \u00a0min\t\r \u00a0at\t\r \u00a04\u00b0C\t\r \u00a0and\t\r \u00a0supernatants\t\r \u00a0were\t\r \u00a0retained.\t\r \u00a0Nonidet\t\r \u00a0P40\t\r \u00a0post\t\r \u00a0nuclear\t\r \u00a0supernatants\t\r \u00a0were\t\r \u00a0immediately\t\r \u00a0placed\t\r \u00a0on\t\r \u00a0ice,\t\r \u00a0while\t\r \u00a0Na\t\r \u00a0deoxycholate\t\r \u00a0and\t\r \u00a0NH4HCO3\t\r \u00a0were\t\r \u00a0added\t\r \u00a0to\t\r \u00a0mechanically\t\r \u00a0lysed\t\r \u00a0post\t\r \u00a0nuclear\t\r \u00a0supernatants\t\r \u00a0to\t\r \u00a0final\t\r \u00a0concentration\t\r \u00a0of\t\r \u00a01%\t\r \u00a0w\/v\t\r \u00a0and\t\r \u00a050\t\r \u00a0mM\t\r \u00a0respectively.\t\r \u00a0Na\t\r \u00a0deoxycholate\t\r \u00a0samples\t\r \u00a0were\t\r \u00a0immediately\t\r \u00a0heated\t\r \u00a0at\t\r \u00a099\u00b0C\t\r \u00a0for\t\r \u00a05\t\r \u00a0min.\t\r \u00a0All\t\r \u00a0samples\t\r \u00a0were\t\r \u00a0then\t\r \u00a0placed\t\r \u00a0on\t\r \u00a0ice\t\r \u00a0or\t\r \u00a0at\t\r \u00a0room\t\r \u00a0temperature\t\r \u00a0as\t\r \u00a0indicated\t\r \u00a0for\t\r \u00a060\t\r \u00a0min.\t\r \u00a0Sample\t\r \u00a0volumes\t\r \u00a0corresponding\t\r \u00a0to\t\r \u00a0100\t\r \u00a0\u03bcg\t\r \u00a0protein\t\r \u00a0(as\t\r \u00a0measured\t\r \u00a0by\t\r \u00a0Bradford\t\r \u00a0Assay)\t\r \u00a0were\t\r \u00a0all\t\r \u00a0adjusted\t\r \u00a0to\t\r \u00a025\t\r \u00a0\u03bcL.\t\r \u00a0Levels\t\r \u00a0of\t\r \u00a0free\t\r \u00a0phosphate\t\r \u00a0in\t\r \u00a0all\t\r \u00a0samples\t\r \u00a0and\t\r \u00a0buffers\t\r \u00a0were\t\r \u00a0assayed\t\r \u00a0exactly\t\r \u00a0as\t\r \u00a0described\t\r \u00a0in\t\r \u00a0the\t\r \u00a0manufacturer\u2019s\t\r \u00a0instructions\t\r \u00a0(Ser\/Thr\t\r \u00a0Phosphatase\t\r \u00a0Assay\t\r \u00a0Kit\t\r \u00a01\t\r \u00a0(K-\u00ad\u2010R-\u00ad\u2010pT-\u00ad\u2010I-\u00ad\u2010R-\u00ad\u2010R),\t\r \u00a0Millipore).\t\r \u00a0Absorbance\t\r \u00a0readings\t\r \u00a0at\t\r \u00a0620\t\r \u00a0nm\t\r \u00a0were\t\r \u00a0determined\t\r \u00a0using\t\r \u00a0a\t\r \u00a0microtiter\t\r \u00a0plate\t\r \u00a0reader\t\r \u00a0(Spectra\t\r \u00a0Fluor\t\r \u00a0Plus,\t\r \u00a0TECAN).\t\r \u00a0\t\r \u00a0 \t\r \u00a0\t\r \u00a0 92\t\r \u00a0 3.2.3\t\r \u00a0Tryptic\t\r \u00a0digest\t\r \u00a0and\t\r \u00a0desalting\t\r \u00a0Protein\t\r \u00a0samples\t\r \u00a0solubilised\t\r \u00a0in\t\r \u00a01%\t\r \u00a0w\/v\t\r \u00a0Na\t\r \u00a0deoxycholate,\t\r \u00a050\t\r \u00a0mM\t\r \u00a0NH4HCO3\t\r \u00a0and\t\r \u00a0heated\t\r \u00a0at\t\r \u00a099\u00b0C\t\r \u00a0for\t\r \u00a05min\t\r \u00a0were\t\r \u00a0reduced,\t\r \u00a0alkylated\t\r \u00a0and\t\r \u00a0digested\t\r \u00a0as\t\r \u00a0previously\t\r \u00a0described\t\r \u00a0(Chapter\t\r \u00a02).\t\r \u00a0For\t\r \u00a0desalting\t\r \u00a0of\t\r \u00a0tryptic\t\r \u00a0peptide\t\r \u00a0samples,\t\r \u00a0a\t\r \u00a0procedure\t\r \u00a0similar\t\r \u00a0to\t\r \u00a0STAGE\t\r \u00a0tips\t\r \u00a0was\t\r \u00a0utilized,\t\r \u00a0excepting\t\r \u00a0that\t\r \u00a0C18\t\r \u00a0packing\t\r \u00a0material\t\r \u00a0was\t\r \u00a0used\t\r \u00a0to\t\r \u00a0increase\t\r \u00a0binding\t\r \u00a0capacity\t\r \u00a0(WP\t\r \u00a0C18\t\r \u00a0Prep\t\r \u00a0HPLC\t\r \u00a0Packing,\t\r \u00a0Baker\t\r \u00a0Analyzed)(358).\t\r \u00a0Briefly,\t\r \u00a0trypsinized\t\r \u00a0samples\t\r \u00a0were\t\r \u00a0diluted\t\r \u00a03x\t\r \u00a0with\t\r \u00a01%\t\r \u00a0v\/v\t\r \u00a0trifluoroacetic\t\r \u00a0acid\t\r \u00a0(TFA),\t\r \u00a05%\t\r \u00a0v\/v\t\r \u00a0CH3CN\t\r \u00a0and\t\r \u00a0clarified\t\r \u00a0at\t\r \u00a016000\t\r \u00a0rcf\t\r \u00a0for\t\r \u00a010\t\r \u00a0min\t\r \u00a0at\t\r \u00a0RT.\t\r \u00a0100\t\r \u00a0\u03bcL\t\r \u00a0of\t\r \u00a0a\t\r \u00a01:1\t\r \u00a0v\/v\t\r \u00a0slurry\t\r \u00a0of\t\r \u00a0C18\t\r \u00a0beads\t\r \u00a0in\t\r \u00a0methanol\t\r \u00a0was\t\r \u00a0added\t\r \u00a0to\t\r \u00a0a\t\r \u00a0STAGE\t\r \u00a0tip.\t\r \u00a0Each\t\r \u00a0tip\t\r \u00a0was\t\r \u00a0first\t\r \u00a0conditioned\t\r \u00a0with\t\r \u00a0100\t\r \u00a0\u03bcL\t\r \u00a0methanol\t\r \u00a0and\t\r \u00a0washed\t\r \u00a0with\t\r \u00a0300\t\r \u00a0\u03bcL\t\r \u00a01%\t\r \u00a0v\/v\t\r \u00a0TFA,\t\r \u00a05%\t\r \u00a0v\/v\t\r \u00a0acetonitrile\t\r \u00a0before\t\r \u00a0the\t\r \u00a0sample\t\r \u00a0was\t\r \u00a0passed\t\r \u00a0through.\t\r \u00a0The\t\r \u00a0binding\t\r \u00a0capacity\t\r \u00a0of\t\r \u00a0each\t\r \u00a0tip\t\r \u00a0for\t\r \u00a0peptides\t\r \u00a0was\t\r \u00a0estimated\t\r \u00a0at\t\r \u00a0500\t\r \u00a0\u03bcg.\t\r \u00a0Tips\t\r \u00a0were\t\r \u00a0washed\t\r \u00a03x\t\r \u00a0with\t\r \u00a0300\t\r \u00a0\u03bcL\t\r \u00a01%\t\r \u00a0v\/v\t\r \u00a0TFA,\t\r \u00a05%\t\r \u00a0v\/v\t\r \u00a0CH3CN\t\r \u00a0and\t\r \u00a0eluted\t\r \u00a0with\t\r \u00a0300\t\r \u00a0\u03bcL\t\r \u00a00.5%\t\r \u00a0v\/v\t\r \u00a0CH3COOH,\t\r \u00a080%\t\r \u00a0v\/v\t\r \u00a0CH3CN.\t\r \u00a0\t\r \u00a0 3.2.4\t\r \u00a0In-\u00ad\u2010solution\t\r \u00a0isoelectric\t\r \u00a0focusing\t\r \u00a0Desalted\t\r \u00a0tryptic\t\r \u00a0peptide\t\r \u00a0samples\t\r \u00a0were\t\r \u00a0dried\t\r \u00a0completely\t\r \u00a0in\t\r \u00a0a\t\r \u00a0vacuum\t\r \u00a0centrifuge\t\r \u00a0(Vacufuge\t\r \u00a0Plus,\t\r \u00a0Eppendorf)\t\r \u00a0and\t\r \u00a0resuspended\t\r \u00a0in\t\r \u00a0a\t\r \u00a05%\t\r \u00a0v\/v\t\r \u00a0glycerol\t\r \u00a0in\t\r \u00a0distilled\t\r \u00a0water\t\r \u00a0containing\t\r \u00a02%\t\r \u00a0w\/v\t\r \u00a0ampholytes\t\r \u00a0of\t\r \u00a0pH\t\r \u00a0range\t\r \u00a03-\u00ad\u201010\t\r \u00a0(Bio-\u00ad\u2010Lyte\t\r \u00a0Ampholyte\t\r \u00a03\/10\t\r \u00a0(BIO-\u00ad\u2010RAD)\t\r \u00a0or\t\r \u00a03-\u00ad\u20106\t\r \u00a0(Fluka\t\r \u00a0Analytical)).\t\r \u00a0Peptide\t\r \u00a0solutions\t\r \u00a0were\t\r \u00a0separated\t\r \u00a0by\t\r \u00a0in-\u00ad\u2010solution\t\r \u00a0isoelectric\t\r \u00a0focusing\t\r \u00a0according\t\r \u00a0to\t\r \u00a0the\t\r \u00a0manufacturers\t\r \u00a0instructions\t\r \u00a0(MicroRotofor\t\r \u00a0Cell,\t\r \u00a0BIO-\u00ad\u2010RAD,\t\r \u00a0referred\t\r \u00a0to\t\r \u00a0as\t\r \u00a0solution-\u00ad\u2010based\t\r \u00a0IEF\t\r \u00a0throughout\t\r \u00a0the\t\r \u00a0text)\t\r \u00a0using\t\r \u00a0a\t\r \u00a0power\t\r \u00a0of\t\r \u00a01.0W\t\r \u00a0until\t\r \u00a0the\t\r \u00a0voltage\t\r \u00a0stabilized\t\r \u00a0(~3\t\r \u00a0h).\t\r \u00a010\t\r \u00a0 \t\r \u00a0\t\r \u00a0 93\t\r \u00a0 fractions\t\r \u00a0were\t\r \u00a0harvested\t\r \u00a0from\t\r \u00a0each\t\r \u00a0IEF\t\r \u00a0run\t\r \u00a0exactly\t\r \u00a0as\t\r \u00a0described\t\r \u00a0in\t\r \u00a0the\t\r \u00a0manufacturer\u2019s\t\r \u00a0instructions.\t\r \u00a0\t\r \u00a0 3.2.5\t\r \u00a0Phosphopeptide\t\r \u00a0enrichment\t\r \u00a0Trypsinized\t\r \u00a0samples\t\r \u00a0or\t\r \u00a0fractions\t\r \u00a0harvested\t\r \u00a0from\t\r \u00a0solution-\u00ad\u2010based\t\r \u00a0IEF\t\r \u00a0were\t\r \u00a0diluted\t\r \u00a03x\t\r \u00a0with\t\r \u00a01%\t\r \u00a0v\/v\t\r \u00a0TFA\t\r \u00a0acid,\t\r \u00a05%\t\r \u00a0v\/v\t\r \u00a0CH3CN,\t\r \u00a0the\t\r \u00a0pH\t\r \u00a0was\t\r \u00a0adjusted\t\r \u00a0to\t\r \u00a0~1,\t\r \u00a0and\t\r \u00a0lactic\t\r \u00a0acid\t\r \u00a0was\t\r \u00a0added\t\r \u00a0to\t\r \u00a0a\t\r \u00a0final\t\r \u00a0concentration\t\r \u00a0of\t\r \u00a0300\t\r \u00a0mg\/mL(412).\t\r \u00a0In\t\r \u00a0parallel,\t\r \u00a010\t\r \u00a0mg\t\r \u00a0of\t\r \u00a0titanium\t\r \u00a0dioxide\t\r \u00a0(TiO2)\t\r \u00a0beads\t\r \u00a0(Titansphere,\t\r \u00a0GL\t\r \u00a0Sciences)\t\r \u00a0for\t\r \u00a0every\t\r \u00a0milligram\t\r \u00a0of\t\r \u00a0starting\t\r \u00a0protein\t\r \u00a0material\t\r \u00a0was\t\r \u00a0washed\t\r \u00a0with\t\r \u00a01mL\t\r \u00a0Solution\t\r \u00a0A\t\r \u00a0(70%\t\r \u00a0v\/v\t\r \u00a0CH3CN,\t\r \u00a00.1%\t\r \u00a0v\/v\t\r \u00a0TFA,\t\r \u00a0300\t\r \u00a0mg\/mL\t\r \u00a0lactic\t\r \u00a0acid).\t\r \u00a0The\t\r \u00a0TiO2\t\r \u00a0beads\t\r \u00a0were\t\r \u00a0incubated\t\r \u00a0with\t\r \u00a0the\t\r \u00a0acidified\t\r \u00a0peptide\t\r \u00a0sample\t\r \u00a0for\t\r \u00a0at\t\r \u00a0least\t\r \u00a01\t\r \u00a0h\t\r \u00a0at\t\r \u00a0RT\t\r \u00a0shaking\t\r \u00a0at\t\r \u00a01400\t\r \u00a0revolutions\t\r \u00a0per\t\r \u00a0minute\t\r \u00a0(rpm).\t\r \u00a0A\t\r \u00a0STAGE\t\r \u00a0tip\t\r \u00a0was\t\r \u00a0constructed\t\r \u00a0as\t\r \u00a0originally\t\r \u00a0described,\t\r \u00a0except\t\r \u00a0that\t\r \u00a0C8\t\r \u00a0material\t\r \u00a0was\t\r \u00a0used\t\r \u00a0as\t\r \u00a0a\t\r \u00a0frit\t\r \u00a0to\t\r \u00a0retain\t\r \u00a0the\t\r \u00a0TiO2\t\r \u00a0beads\t\r \u00a0(C8\t\r \u00a0Empore\t\r \u00a0Disks,\t\r \u00a03M)(358).\t\r \u00a0The\t\r \u00a0C8\t\r \u00a0STAGE\t\r \u00a0tip\t\r \u00a0was\t\r \u00a0washed\t\r \u00a0with\t\r \u00a0Solution\t\r \u00a0A\t\r \u00a0and\t\r \u00a0the\t\r \u00a0sample\t\r \u00a0was\t\r \u00a0passed\t\r \u00a0through\t\r \u00a0by\t\r \u00a0centrifugation\t\r \u00a0at\t\r \u00a01000\t\r \u00a0rcf\t\r \u00a0at\t\r \u00a0RT.\t\r \u00a0Similarly\t\r \u00a0the\t\r \u00a0tip\t\r \u00a0was\t\r \u00a0washed\t\r \u00a0at\t\r \u00a0least\t\r \u00a05x\t\r \u00a0with\t\r \u00a0300\t\r \u00a0\u03bcL\t\r \u00a0Solution\t\r \u00a0A\t\r \u00a0and\t\r \u00a0subsequently\t\r \u00a05x\t\r \u00a0with\t\r \u00a0300\t\r \u00a0\u03bcL\t\r \u00a0Solution\t\r \u00a0B\t\r \u00a0(80%\t\r \u00a0v\/v\t\r \u00a0CH3CN,\t\r \u00a00.1%\t\r \u00a0v\/v\t\r \u00a0TFA).\t\r \u00a0Peptides\t\r \u00a0were\t\r \u00a0eluted\t\r \u00a0stepwise\t\r \u00a0with\t\r \u00a050\t\r \u00a0\u03bcL\t\r \u00a00.5%\t\r \u00a0v\/v\t\r \u00a0NH4OH,\t\r \u00a0followed\t\r \u00a0by\t\r \u00a050\t\r \u00a0\u03bcL\t\r \u00a00.5%\t\r \u00a0v\/v\t\r \u00a0NH4OH,\t\r \u00a030%\t\r \u00a0v\/v\t\r \u00a0CH3CN\t\r \u00a0and\t\r \u00a050\t\r \u00a0\u03bcL\t\r \u00a00.5%\t\r \u00a0v\/v\t\r \u00a0NH4OH,\t\r \u00a050%\t\r \u00a0v\/v\t\r \u00a0CH3CN.\t\r \u00a0CH3CN\t\r \u00a0was\t\r \u00a0diluted\t\r \u00a0from\t\r \u00a0the\t\r \u00a0eluate\t\r \u00a0to\t\r \u00a0<10%\t\r \u00a0with\t\r \u00a01%\t\r \u00a0v\/v\t\r \u00a0TFA\t\r \u00a0acid,\t\r \u00a05%\t\r \u00a0v\/v\t\r \u00a0CH3CN\t\r \u00a0and\t\r \u00a0the\t\r \u00a0sample\t\r \u00a0was\t\r \u00a0concentrated\t\r \u00a0on\t\r \u00a0a\t\r \u00a0STAGE\t\r \u00a0tip\t\r \u00a0as\t\r \u00a0previously\t\r \u00a0described(490).\t\r \u00a0\t\r \u00a0 \t\r \u00a0\t\r \u00a0 94\t\r \u00a0 3.2.6\t\r \u00a0Testing\t\r \u00a0interference\t\r \u00a0of\t\r \u00a0phosphopeptide\t\r \u00a0enrichment\t\r \u00a0by\t\r \u00a0phosphatase\t\r \u00a0 inhibitors\t\r \u00a0Confluent,\t\r \u00a010\t\r \u00a0cm\t\r \u00a0plates\t\r \u00a0of\t\r \u00a0HeLa\t\r \u00a0cells\t\r \u00a0were\t\r \u00a0cultured\t\r \u00a0as\t\r \u00a0described\t\r \u00a0above.\t\r \u00a0Cells\t\r \u00a0were\t\r \u00a0washed\t\r \u00a03x\t\r \u00a0with\t\r \u00a0PBS,\t\r \u00a0harvested\t\r \u00a0with\t\r \u00a0a\t\r \u00a0scraper,\t\r \u00a0and\t\r \u00a0pelleted\t\r \u00a0at\t\r \u00a0600\t\r \u00a0rcf\t\r \u00a0for\t\r \u00a05\t\r \u00a0min\t\r \u00a0at\t\r \u00a04\u00b0C.\t\r \u00a0Cell\t\r \u00a0pellets\t\r \u00a0corresponding\t\r \u00a0to\t\r \u00a0one\t\r \u00a010\t\r \u00a0cm\t\r \u00a0plates\t\r \u00a0were\t\r \u00a0resuspended\t\r \u00a0in\t\r \u00a0200\t\r \u00a0\u03bcL\t\r \u00a0of\t\r \u00a0the\t\r \u00a0following\t\r \u00a0lysis\t\r \u00a0buffers\t\r \u00a0and\t\r \u00a0used\t\r \u00a0for\t\r \u00a0each\t\r \u00a0of\t\r \u00a0the\t\r \u00a06\t\r \u00a0conditions\t\r \u00a0described.\t\r \u00a0For\t\r \u00a0the\t\r \u00a0\u2018All\t\r \u00a0inhibitors\u2019\t\r \u00a0and\t\r \u00a0\u2018Desalting\u2019\t\r \u00a0conditions,\t\r \u00a0cells\t\r \u00a0were\t\r \u00a0resuspended\t\r \u00a0in\t\r \u00a01xPBS,\t\r \u00a01\t\r \u00a0mM\t\r \u00a0Na3VO4,\t\r \u00a05\t\r \u00a0mM\t\r \u00a0sodium\t\r \u00a0pyrophosphate,\t\r \u00a010\t\r \u00a0mM\t\r \u00a0NaF,\t\r \u00a020\t\r \u00a0mM\t\r \u00a0\u03b2-\u00ad\u2010glycerophosphate,\t\r \u00a00.5\t\r \u00a0mM\t\r \u00a0pervanadate,\t\r \u00a010\t\r \u00a0\u03bcM\t\r \u00a0deltamethrin,\t\r \u00a0100\t\r \u00a0nM\t\r \u00a0calyculin\t\r \u00a0A,\t\r \u00a0and\t\r \u00a0protease\t\r \u00a0inhibitor\t\r \u00a0cocktail\t\r \u00a0(Complete,\t\r \u00a0Roche\t\r \u00a0Diagnostics).\t\r \u00a0Each\t\r \u00a0of\t\r \u00a0the\t\r \u00a0other\t\r \u00a0conditions\t\r \u00a0were\t\r \u00a0identical\t\r \u00a0except\t\r \u00a0for\t\r \u00a0the\t\r \u00a0exclusion\t\r \u00a0of\t\r \u00a0the\t\r \u00a0indicated\t\r \u00a0phosphatase\t\r \u00a0inhibitor(s).\t\r \u00a0Cells\t\r \u00a0were\t\r \u00a0lysed\t\r \u00a0mechanically\t\r \u00a0by\t\r \u00a05-\u00ad\u20106\t\r \u00a0passages\t\r \u00a0through\t\r \u00a0a\t\r \u00a022G\t\r \u00a0needle,\t\r \u00a0and\t\r \u00a0nuclei\t\r \u00a0were\t\r \u00a0pelleted\t\r \u00a0at\t\r \u00a0600\t\r \u00a0rcf\t\r \u00a0for\t\r \u00a05\t\r \u00a0min\t\r \u00a0at\t\r \u00a04\u00b0C.\t\r \u00a0Post\t\r \u00a0nuclear\t\r \u00a0supernatants\t\r \u00a0were\t\r \u00a0retained\t\r \u00a0and\t\r \u00a0Na\t\r \u00a0deoxycholate\t\r \u00a0and\t\r \u00a0NH4HCO3\t\r \u00a0were\t\r \u00a0immediately\t\r \u00a0added\t\r \u00a0to\t\r \u00a0final\t\r \u00a0concentrations\t\r \u00a0of\t\r \u00a01%\t\r \u00a0w\/v\t\r \u00a0and\t\r \u00a050\t\r \u00a0mM\t\r \u00a0respectively.\t\r \u00a0Samples\t\r \u00a0were\t\r \u00a0immediately\t\r \u00a0placed\t\r \u00a0on\t\r \u00a0a\t\r \u00a0heating\t\r \u00a0block\t\r \u00a0at\t\r \u00a099\u00b0C\t\r \u00a0for\t\r \u00a05\t\r \u00a0min,\t\r \u00a0cooled\t\r \u00a0to\t\r \u00a0RT.\t\r \u00a0All\t\r \u00a0samples\t\r \u00a0were\t\r \u00a0then\t\r \u00a0subjected\t\r \u00a0to\t\r \u00a0an\t\r \u00a0in-\u00ad\u2010solution\t\r \u00a0digest\t\r \u00a0as\t\r \u00a0described\t\r \u00a0above.\t\r \u00a0For\t\r \u00a0the\t\r \u00a0\u2018Desalting\u2019\t\r \u00a0condition,\t\r \u00a0tryptic\t\r \u00a0peptide\t\r \u00a0samples\t\r \u00a0were\t\r \u00a0desalted\t\r \u00a0as\t\r \u00a0described\t\r \u00a0above\t\r \u00a0and\t\r \u00a0phosphopeptide\t\r \u00a0enrichment\t\r \u00a0was\t\r \u00a0performed\t\r \u00a0exactly\t\r \u00a0as\t\r \u00a0described\t\r \u00a0above.\t\r \u00a0\t\r \u00a0 3.2.7\t\r \u00a0Mass\t\r \u00a0spectrometry\t\r \u00a0For\t\r \u00a0LC-\u00ad\u2010MSn,\t\r \u00a0samples\t\r \u00a0were\t\r \u00a0analysed\t\r \u00a0on\t\r \u00a0a\t\r \u00a0LTQ-\u00ad\u2010OrbitrapXL\t\r \u00a0(Thermo\t\r \u00a0Fisher\t\r \u00a0Scientific)\t\r \u00a0coupled\t\r \u00a0on-\u00ad\u2010line\t\r \u00a0to\t\r \u00a0an\t\r \u00a0Agilent\t\r \u00a01100\t\r \u00a0Series\t\r \u00a0nanoflow\t\r \u00a0HPLC\t\r \u00a0instrument\t\r \u00a0 \t\r \u00a0\t\r \u00a0 95\t\r \u00a0 using\t\r \u00a0a\t\r \u00a0nanospray\t\r \u00a0ionization\t\r \u00a0source\t\r \u00a0(Proxeon\t\r \u00a0Biosystems)\t\r \u00a0as\t\r \u00a0previously\t\r \u00a0described\t\r \u00a0(Chapter\t\r \u00a02).\t\r \u00a0For\t\r \u00a0conditions\t\r \u00a0utilizing\t\r \u00a0MultiStage\t\r \u00a0Activation,\t\r \u00a0neutral\t\r \u00a0loss\t\r \u00a0masses\t\r \u00a0of\t\r \u00a033\t\r \u00a0dalton\t\r \u00a0(Da),\t\r \u00a049\t\r \u00a0Da,\t\r \u00a0and\t\r \u00a098\t\r \u00a0Da\t\r \u00a0were\t\r \u00a0selected\t\r \u00a0when\t\r \u00a0within\t\r \u00a0the\t\r \u00a0top\t\r \u00a0n=3.\t\r \u00a0For\t\r \u00a0conditions\t\r \u00a0including\t\r \u00a0singly\t\r \u00a0charged\t\r \u00a0peptides,\t\r \u00a0charge\t\r \u00a0state\t\r \u00a0rejection\t\r \u00a0was\t\r \u00a0only\t\r \u00a0enabled\t\r \u00a0for\t\r \u00a0cases\t\r \u00a0of\t\r \u00a0unassigned\t\r \u00a0charge\t\r \u00a0states.\t\r \u00a0Monoisotopic\t\r \u00a0peaks\t\r \u00a0and\t\r \u00a0charge\t\r \u00a0states\t\r \u00a0in\t\r \u00a0the\t\r \u00a0data\t\r \u00a0were\t\r \u00a0extracted\t\r \u00a0and\t\r \u00a0corrected\t\r \u00a0using\t\r \u00a0DTA\t\r \u00a0Supercharge\t\r \u00a0(http:\/\/msquant.sourceforge.net\/),\t\r \u00a0and\t\r \u00a0peak\t\r \u00a0lists\t\r \u00a0were\t\r \u00a0searched\t\r \u00a0against\t\r \u00a0a\t\r \u00a0database\t\r \u00a0containing\t\r \u00a0all\t\r \u00a0human\t\r \u00a0protein\t\r \u00a0sequences\t\r \u00a0in\t\r \u00a0the\t\r \u00a0International\t\r \u00a0Proteome\t\r \u00a0Index\t\r \u00a0using\t\r \u00a0Mascot\t\r \u00a0(v2.2,\t\r \u00a0Matrix\t\r \u00a0Science).\t\r \u00a0Search\t\r \u00a0parameters\t\r \u00a0included\t\r \u00a0one\t\r \u00a0missed\t\r \u00a0cleavage,\t\r \u00a0cysteine\t\r \u00a0carbamidomethyl\t\r \u00a0fixed\t\r \u00a0modification,\t\r \u00a0and\t\r \u00a0variable\t\r \u00a0modifications\t\r \u00a0methionine\t\r \u00a0oxidation\t\r \u00a0and\t\r \u00a0phosphorylation\t\r \u00a0on\t\r \u00a0serine,\t\r \u00a0threonine,\t\r \u00a0and\t\r \u00a0tyrosine\t\r \u00a0residues.\t\r \u00a0The\t\r \u00a0peptide\t\r \u00a0mass\t\r \u00a0tolerance\t\r \u00a0was\t\r \u00a010\t\r \u00a0parts-\u00ad\u2010per-\u00ad\u2010million\t\r \u00a0and\t\r \u00a0the\t\r \u00a0MS\/MS\t\r \u00a0tolerance\t\r \u00a0was\t\r \u00a00.6\t\r \u00a0Da.\t\r \u00a0MSQuant\t\r \u00a0v1.5b7(350)\t\r \u00a0was\t\r \u00a0used\t\r \u00a0for\t\r \u00a0parsing\t\r \u00a0Mascot\t\r \u00a0result\t\r \u00a0files,\t\r \u00a0iterative\t\r \u00a0mass\t\r \u00a0recalibration,\t\r \u00a0and\t\r \u00a0assigning\t\r \u00a0PTM\t\r \u00a0scores.\t\r \u00a0\t\r \u00a0 3.3\t\r \u00a0Results\t\r \u00a0 3.3.1\t\r \u00a0Phosphatase\t\r \u00a0inhibitors\t\r \u00a0versus\t\r \u00a0heat\/chaotropic\t\r \u00a0denaturation\t\r \u00a0for\t\r \u00a0 minimizing\t\r \u00a0phosphatase\t\r \u00a0activity\t\r \u00a0Phosphatases\t\r \u00a0are\t\r \u00a0robust,\t\r \u00a0highly\t\r \u00a0efficient\t\r \u00a0enzymes\t\r \u00a0that\t\r \u00a0exhibit\t\r \u00a0much\t\r \u00a0less\t\r \u00a0specificity\t\r \u00a0than\t\r \u00a0kinases\t\r \u00a0and\t\r \u00a0will\t\r \u00a0be\t\r \u00a0active\t\r \u00a0in\t\r \u00a0most\t\r \u00a0cell\t\r \u00a0lysis\t\r \u00a0procedures.\t\r \u00a0Thus,\t\r \u00a0their\t\r \u00a0acitivity\t\r \u00a0must\t\r \u00a0be\t\r \u00a0minimized\t\r \u00a0or\t\r \u00a0eliminated\t\r \u00a0if\t\r \u00a0one\t\r \u00a0wishes\t\r \u00a0to\t\r \u00a0study\t\r \u00a0the\t\r \u00a0phosphorylated\t\r \u00a0state\t\r \u00a0of\t\r \u00a0proteins.\t\r \u00a0Biochemists\t\r \u00a0have\t\r \u00a0typically\t\r \u00a0used\t\r \u00a0broad-\u00ad\u2010spectrum,\t\r \u00a0competitive\t\r \u00a0inhibitors\t\r \u00a0of\t\r \u00a0protein\t\r \u00a0phosphatases\t\r \u00a0to\t\r \u00a0preserve\t\r \u00a0phosphorylations,\t\r \u00a0but\t\r \u00a0recent\t\r \u00a0 \t\r \u00a0\t\r \u00a0 96\t\r \u00a0 evidence\t\r \u00a0suggests\t\r \u00a0that\t\r \u00a0this\t\r \u00a0classical\t\r \u00a0approach\t\r \u00a0is\t\r \u00a0not\t\r \u00a0as\t\r \u00a0effective\t\r \u00a0as\t\r \u00a0one\t\r \u00a0would\t\r \u00a0hope(387,\t\r \u00a0388).\t\r \u00a0Besides\t\r \u00a0inhibitors,\t\r \u00a0another\t\r \u00a0approach\t\r \u00a0for\t\r \u00a0eliminating\t\r \u00a0phosphatase\t\r \u00a0activity\t\r \u00a0in\t\r \u00a0a\t\r \u00a0cell\t\r \u00a0lysate\t\r \u00a0is\t\r \u00a0to\t\r \u00a0quickly\t\r \u00a0and\t\r \u00a0completely\t\r \u00a0denature\t\r \u00a0all\t\r \u00a0proteins\t\r \u00a0in\t\r \u00a0the\t\r \u00a0sample.\t\r \u00a0Heat\t\r \u00a0denaturation\t\r \u00a0alone\t\r \u00a0in\t\r \u00a0the\t\r \u00a0absence\t\r \u00a0of\t\r \u00a0a\t\r \u00a0chaotropic\t\r \u00a0surfactant\t\r \u00a0is\t\r \u00a0effective\t\r \u00a0for\t\r \u00a0eliminating\t\r \u00a0phosphatase\t\r \u00a0activity\t\r \u00a0but\t\r \u00a0often\t\r \u00a0results\t\r \u00a0in\t\r \u00a0precipitates\t\r \u00a0that\t\r \u00a0are\t\r \u00a0difficult\t\r \u00a0to\t\r \u00a0resolubilize\t\r \u00a0(results\t\r \u00a0not\t\r \u00a0shown).\t\r \u00a0SDS\t\r \u00a0is,\t\r \u00a0of\t\r \u00a0course,\t\r \u00a0a\t\r \u00a0very\t\r \u00a0effective\t\r \u00a0chaotrope\t\r \u00a0but\t\r \u00a0must\t\r \u00a0be\t\r \u00a0removed\t\r \u00a0(i.e.\t\r \u00a0by\t\r \u00a0filtration)\t\r \u00a0prior\t\r \u00a0to\t\r \u00a0tryptic\t\r \u00a0digestion\t\r \u00a0and\t\r \u00a0LC-\u00ad\u2010MSn(491,\t\r \u00a0492).\t\r \u00a0Alternatively,\t\r \u00a0deoxycholate\t\r \u00a0is\t\r \u00a0also\t\r \u00a0an\t\r \u00a0effective\t\r \u00a0chaotrope\t\r \u00a0and,\t\r \u00a0when\t\r \u00a0used\t\r \u00a0at\t\r \u00a00.5-\u00ad\u20101%\t\r \u00a0w\/v,\t\r \u00a0greatly\t\r \u00a0improves\t\r \u00a0protein\t\r \u00a0solubility\t\r \u00a0without\t\r \u00a0interfering\t\r \u00a0with\t\r \u00a0tryptic\t\r \u00a0digestion(493,\t\r \u00a0494).\t\r \u00a0After\t\r \u00a0acidifying\t\r \u00a0the\t\r \u00a0sample\t\r \u00a0to\t\r \u00a0pH<4\t\r \u00a0it\t\r \u00a0can\t\r \u00a0be\t\r \u00a0quantitatively\t\r \u00a0eliminated\t\r \u00a0either\t\r \u00a0by\t\r \u00a0precipitation\t\r \u00a0or\t\r \u00a0transfer\t\r \u00a0to\t\r \u00a0an\t\r \u00a0organic\t\r \u00a0phase\t\r \u00a0prior\t\r \u00a0to\t\r \u00a0LC-\u00ad\u2010MSn.\t\r \u00a0When\t\r \u00a0we\t\r \u00a0compared\t\r \u00a0free\t\r \u00a0phosphate\t\r \u00a0levels\t\r \u00a0in\t\r \u00a0a\t\r \u00a0post-\u00ad\u2010nuclear\t\r \u00a0supernatant\t\r \u00a0prepared\t\r \u00a0with\t\r \u00a0conventional\t\r \u00a0phosphatase\t\r \u00a0inhibitors\t\r \u00a0to\t\r \u00a0a\t\r \u00a0similar\t\r \u00a0post-\u00ad\u2010nuclear\t\r \u00a0supernatant\t\r \u00a0treated\t\r \u00a0with\t\r \u00a01%\t\r \u00a0w\/v\t\r \u00a0deoxycholate\t\r \u00a0and\t\r \u00a0heated\t\r \u00a0to\t\r \u00a099\u00b0C,\t\r \u00a0the\t\r \u00a0deoxycholate\t\r \u00a0treated\t\r \u00a0condition\t\r \u00a0was\t\r \u00a0very\t\r \u00a0clearly\t\r \u00a0more\t\r \u00a0effective\t\r \u00a0at\t\r \u00a0preventing\t\r \u00a0the\t\r \u00a0release\t\r \u00a0of\t\r \u00a0free\t\r \u00a0phosphate\t\r \u00a0during\t\r \u00a0a\t\r \u00a01\t\r \u00a0h\t\r \u00a0incubation\t\r \u00a0either\t\r \u00a0on\t\r \u00a0ice\t\r \u00a0or\t\r \u00a0at\t\r \u00a0RT\t\r \u00a0(Figure\t\r \u00a03.1A).\t\r \u00a0In\t\r \u00a0addition,\t\r \u00a0when\t\r \u00a0conventional\t\r \u00a0inhibitors\t\r \u00a0were\t\r \u00a0used\t\r \u00a0the\t\r \u00a0amount\t\r \u00a0of\t\r \u00a0detected\t\r \u00a0phosphate\t\r \u00a0increased\t\r \u00a0dramatically\t\r \u00a0between\t\r \u00a0the\t\r \u00a0incubation\t\r \u00a0on\t\r \u00a0ice\t\r \u00a0and\t\r \u00a0at\t\r \u00a0RT,\t\r \u00a0whereas\t\r \u00a0when\t\r \u00a0deoxycholate\/denaturation\t\r \u00a0was\t\r \u00a0used\t\r \u00a0this\t\r \u00a0difference\t\r \u00a0was\t\r \u00a0minimal.\t\r \u00a0The\t\r \u00a0number\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0identified\t\r \u00a0using\t\r \u00a0deoxycholate\t\r \u00a0alone\t\r \u00a0or\t\r \u00a0in\t\r \u00a0combination\t\r \u00a0with\t\r \u00a0a\t\r \u00a0cocktail\t\r \u00a0of\t\r \u00a0phosphatase\t\r \u00a0inhibitors\t\r \u00a0was\t\r \u00a0also\t\r \u00a0considered.\t\r \u00a0However\t\r \u00a0adding\t\r \u00a0phosphatase\t\r \u00a0inhibitors\t\r \u00a0offered\t\r \u00a0no\t\r \u00a0improvement\t\r \u00a0over\t\r \u00a0the\t\r \u00a0use\t\r \u00a0of\t\r \u00a0deoxycholate\t\r \u00a0alone\t\r \u00a0(Figure\t\r \u00a03.1B).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 97\t\r \u00a0 The\t\r \u00a0presence\t\r \u00a0of\t\r \u00a0phosphatase\t\r \u00a0inhibitors\t\r \u00a0during\t\r \u00a0phosphopeptide\t\r \u00a0enrichment\t\r \u00a0has\t\r \u00a0one\t\r \u00a0further\t\r \u00a0disadvantage:\t\r \u00a0since\t\r \u00a0many\t\r \u00a0phosphatase\t\r \u00a0inhibitors\t\r \u00a0work\t\r \u00a0by\t\r \u00a0mimicking\t\r \u00a0phosphate\t\r \u00a0groups,\t\r \u00a0they\t\r \u00a0also\t\r \u00a0compete\t\r \u00a0with\t\r \u00a0phosphopeptides\t\r \u00a0for\t\r \u00a0binding\t\r \u00a0to\t\r \u00a0TiO2.\t\r \u00a0Indeed,\t\r \u00a0when\t\r \u00a0a\t\r \u00a0full\t\r \u00a0cocktail\t\r \u00a0of\t\r \u00a0phosphatase\t\r \u00a0inhibitors\t\r \u00a0was\t\r \u00a0present\t\r \u00a0during\t\r \u00a0enrichment\t\r \u00a0by\t\r \u00a0MOC,\t\r \u00a0our\t\r \u00a0phosphopeptide\t\r \u00a0yield\t\r \u00a0was\t\r \u00a0very\t\r \u00a0low\t\r \u00a0(Figure\t\r \u00a03.1C)\t\r \u00a0and\t\r \u00a0eliminating\t\r \u00a0any\t\r \u00a0one\t\r \u00a0of\t\r \u00a0\u00df-\u00ad\u2010glycerophosphate,\t\r \u00a0calyculin\t\r \u00a0A\t\r \u00a0or\t\r \u00a0pyrophosphate\t\r \u00a0did\t\r \u00a0not\t\r \u00a0significantly\t\r \u00a0improve\t\r \u00a0the\t\r \u00a0number\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0identified.\t\r \u00a0Only\t\r \u00a0by\t\r \u00a0eliminating\t\r \u00a0all\t\r \u00a0three\t\r \u00a0phosphomimetic\t\r \u00a0inhibitors\t\r \u00a0were\t\r \u00a0we\t\r \u00a0able\t\r \u00a0to\t\r \u00a0improve\t\r \u00a0the\t\r \u00a0phosphopeptide\t\r \u00a0yield\t\r \u00a0to\t\r \u00a0near\t\r \u00a0the\t\r \u00a0levels\t\r \u00a0obtained\t\r \u00a0by\t\r \u00a0a\t\r \u00a0condition\t\r \u00a0where\t\r \u00a0all\t\r \u00a0the\t\r \u00a0inhibitors\t\r \u00a0were\t\r \u00a0used\t\r \u00a0during\t\r \u00a0cell\t\r \u00a0lysis\t\r \u00a0and\t\r \u00a0then\t\r \u00a0depleted\t\r \u00a0by\t\r \u00a0C18\t\r \u00a0chromatography\t\r \u00a0(desalting)\t\r \u00a0prior\t\r \u00a0to\t\r \u00a0phosphopeptide\t\r \u00a0enrichment.\t\r \u00a0 \t\r \u00a0\t\r \u00a0 98\t\r \u00a0 Figure\t\r \u00a03.1\t\r \u00a0Effects\t\r \u00a0of\t\r \u00a0phosphatase\t\r \u00a0inhibitors\t\r \u00a0during\t\r \u00a0cell\t\r \u00a0lysis\t\r \u00a0and\t\r \u00a0phosphopeptide\t\r \u00a0enrichment\t\r \u00a0 \t\r \u00a0A)\t\r \u00a0Post\t\r \u00a0nuclear\t\r \u00a0supernatants\t\r \u00a0prepared\t\r \u00a0as\t\r \u00a0described\t\r \u00a0in\t\r \u00a0Experimental\t\r \u00a0Procedures\t\r \u00a0were\t\r \u00a0incubated\t\r \u00a0on\t\r \u00a0ice\t\r \u00a0or\t\r \u00a0at\t\r \u00a0RT\t\r \u00a0for\t\r \u00a060\t\r \u00a0min\t\r \u00a0before\t\r \u00a0phosphate\t\r \u00a0levels\t\r \u00a0were\t\r \u00a0measured.\t\r \u00a0Each\t\r \u00a0bar\t\r \u00a0represents\t\r \u00a0the\t\r \u00a0average\t\r \u00a0(+\/-\u00ad\u2010\t\r \u00a0standard\t\r \u00a0deviation)\t\r \u00a0level\t\r \u00a0of\t\r \u00a0free\t\r \u00a0phosphate\t\r \u00a0measured\t\r \u00a0for\t\r \u00a0the\t\r \u00a0described\t\r \u00a0condition\t\r \u00a0after\t\r \u00a0subtraction\t\r \u00a0of\t\r \u00a0individual\t\r \u00a0buffer\t\r \u00a0controls.\t\r \u00a0B)\t\r \u00a0Average\t\r \u00a0(+\/-\u00ad\u2010\t\r \u00a0standard\t\r \u00a0deviation)\t\r \u00a0number\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0identified\t\r \u00a0from\t\r \u00a0cells\t\r \u00a0lysed\t\r \u00a0in\t\r \u00a0deoxycholate\t\r \u00a0with\t\r \u00a0or\t\r \u00a0without\t\r \u00a0inhibitors\t\r \u00a0present.\t\r \u00a0C)\t\r \u00a0Average\t\r \u00a0(+\/-\u00ad\u2010\t\r \u00a0standard\t\r \u00a0deviation)\t\r \u00a0number\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0identified\t\r \u00a0when\t\r \u00a0each\t\r \u00a0or\t\r \u00a0all\t\r \u00a0phosphomimetic\t\r \u00a0inhibitors\t\r \u00a0are\t\r \u00a0absent.\t\r \u00a0\u03b2-\u00ad\u2010glycerophosphate\t\r \u00a0-\u00ad\u2010\t\r \u00a0\u03b2-\u00ad\u2010GP;\t\r \u00a0Na\t\r \u00a0pyrophosphate\t\r \u00a0-\u00ad\u2010\t\r \u00a0NaPPi.\t\r \u00a0\t\r \u00a0 \t\r \u00a0\t\r \u00a0 99\t\r \u00a0 3.3.2\t\r \u00a0Phosphopeptide\t\r \u00a0prefractionation\t\r \u00a0using\t\r \u00a0solution-\u00ad\u2010based\t\r \u00a0isoelectric\t\r \u00a0 focusing\t\r \u00a0It\t\r \u00a0is\t\r \u00a0now\t\r \u00a0widely\t\r \u00a0recognized\t\r \u00a0that\t\r \u00a0prefractionating\t\r \u00a0complex\t\r \u00a0proteomes\t\r \u00a0prior\t\r \u00a0to\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0analysis\t\r \u00a0allows\t\r \u00a0deeper\t\r \u00a0coverage\t\r \u00a0of\t\r \u00a0the\t\r \u00a0components\t\r \u00a0of\t\r \u00a0that\t\r \u00a0sample(294,\t\r \u00a0356).\t\r \u00a0SCX\t\r \u00a0has\t\r \u00a0been\t\r \u00a0used\t\r \u00a0for\t\r \u00a0this\t\r \u00a0purpose\t\r \u00a0previously(390,\t\r \u00a0495)\t\r \u00a0but\t\r \u00a0IEF\t\r \u00a0offers\t\r \u00a0similar\t\r \u00a0separation\t\r \u00a0properties\t\r \u00a0and\t\r \u00a0potentially\t\r \u00a0very\t\r \u00a0high\t\r \u00a0loading\t\r \u00a0capacities.\t\r \u00a0Thus,\t\r \u00a0we\t\r \u00a0examined\t\r \u00a0the\t\r \u00a0utility\t\r \u00a0of\t\r \u00a0IEF\t\r \u00a0in\t\r \u00a0this\t\r \u00a0regard.\t\r \u00a0Using\t\r \u00a0a\t\r \u00a0completely\t\r \u00a0solution-\u00ad\u2010based\t\r \u00a0IEF\t\r \u00a0instrument,\t\r \u00a0we\t\r \u00a0were\t\r \u00a0able\t\r \u00a0to\t\r \u00a0load\t\r \u00a0up\t\r \u00a0to\t\r \u00a030\t\r \u00a0mg\t\r \u00a0of\t\r \u00a0digested\t\r \u00a0total\t\r \u00a0protein\t\r \u00a0and\t\r \u00a0still\t\r \u00a0obtain\t\r \u00a0satisfactory\t\r \u00a0resolution\t\r \u00a0as,\t\r \u00a0under\t\r \u00a0these\t\r \u00a0conditions,\t\r \u00a060%\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0were\t\r \u00a0identified\t\r \u00a0in\t\r \u00a0only\t\r \u00a0one\t\r \u00a0of\t\r \u00a0the\t\r \u00a010\t\r \u00a0fractions\t\r \u00a0collected\t\r \u00a0and\t\r \u00a0more\t\r \u00a0than\t\r \u00a085%\t\r \u00a0were\t\r \u00a0in\t\r \u00a0two\t\r \u00a0or\t\r \u00a0fewer\t\r \u00a0fractions\t\r \u00a0(Figure\t\r \u00a03.2A).\t\r \u00a0As\t\r \u00a0expected,\t\r \u00a0most\t\r \u00a0phosphopeptides\t\r \u00a0have\t\r \u00a0a\t\r \u00a0low\t\r \u00a0isoelectric\t\r \u00a0point\t\r \u00a0(pI)\t\r \u00a0so\t\r \u00a0an\t\r \u00a0ampholyte\t\r \u00a0mix\t\r \u00a0with\t\r \u00a0a\t\r \u00a0pH\t\r \u00a0range\t\r \u00a0between\t\r \u00a03\t\r \u00a0and\t\r \u00a010\t\r \u00a0forced\t\r \u00a0most\t\r \u00a0of\t\r \u00a0the\t\r \u00a0phosphopeptides\t\r \u00a0into\t\r \u00a0the\t\r \u00a0first\t\r \u00a0five\t\r \u00a0fractions,\t\r \u00a0effectively\t\r \u00a0enriching\t\r \u00a0them\t\r \u00a0away\t\r \u00a0from\t\r \u00a0a\t\r \u00a0significant\t\r \u00a0portion\t\r \u00a0of\t\r \u00a0the\t\r \u00a0non-\u00ad\u2010phosphopeptides\t\r \u00a0found\t\r \u00a0at\t\r \u00a0higher\t\r \u00a0pIs(362).\t\r \u00a0This\t\r \u00a0compression\t\r \u00a0could\t\r \u00a0be\t\r \u00a0overcome\t\r \u00a0by\t\r \u00a0either\t\r \u00a0a\t\r \u00a0second\t\r \u00a0round\t\r \u00a0of\t\r \u00a0fractionation\t\r \u00a0where\t\r \u00a0the\t\r \u00a0first\t\r \u00a0five\t\r \u00a0fractions\t\r \u00a0are\t\r \u00a0pooled\t\r \u00a0and\t\r \u00a0and\t\r \u00a0re-\u00ad\u2010fractionated,\t\r \u00a0or\t\r \u00a0by\t\r \u00a0a\t\r \u00a0single\t\r \u00a0fractionation\t\r \u00a0run\t\r \u00a0using\t\r \u00a0an\t\r \u00a0ampholyte\t\r \u00a0mixture\t\r \u00a0ranging\t\r \u00a0in\t\r \u00a0pH\t\r \u00a0from\t\r \u00a03\t\r \u00a0to\t\r \u00a06\t\r \u00a0(Figure.\t\r \u00a03.2B).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 100\t\r \u00a0 Figure\t\r \u00a03.2\t\r \u00a0Phosphopeptide\t\r \u00a0enrichment\t\r \u00a0by\t\r \u00a0in-\u00ad\u2010solution\t\r \u00a0isoelectric\t\r \u00a0focusing\t\r \u00a0 \t\r \u00a0Tryptic\t\r \u00a0peptides\t\r \u00a0from\t\r \u00a0a\t\r \u00a0HeLa\t\r \u00a0cell\t\r \u00a0lysate\t\r \u00a0were\t\r \u00a0separated\t\r \u00a0by\t\r \u00a0IEF.\t\r \u00a0Subsequently\t\r \u00a0phosphopeptides\t\r \u00a0were\t\r \u00a0enriched\t\r \u00a0and\t\r \u00a0analysed\t\r \u00a0by\t\r \u00a0TiO2\t\r \u00a0chromatography\t\r \u00a0and\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0as\t\r \u00a0described.\t\r \u00a0Plotted\t\r \u00a0are\t\r \u00a0a)\t\r \u00a0the\t\r \u00a0number\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0identified\t\r \u00a0in\t\r \u00a0a\t\r \u00a0single\t\r \u00a0versus\t\r \u00a0multiple\t\r \u00a0IEF\t\r \u00a0fractions,\t\r \u00a0and\t\r \u00a0b)\t\r \u00a0the\t\r \u00a0number\t\r \u00a0of\t\r \u00a0peptides\t\r \u00a0found\t\r \u00a0in\t\r \u00a0each\t\r \u00a0fraction.\t\r \u00a0In\t\r \u00a0a)\t\r \u00a0the\t\r \u00a0data\t\r \u00a0represents\t\r \u00a0peptide\t\r \u00a0fractionation\t\r \u00a0using\t\r \u00a0ampholytes\t\r \u00a0within\t\r \u00a0a\t\r \u00a0pH\t\r \u00a0range\t\r \u00a0of\t\r \u00a03-\u00ad\u20106.\t\r \u00a0In\t\r \u00a0b)\t\r \u00a0the\t\r \u00a0dashed\t\r \u00a0curve\t\r \u00a0represents\t\r \u00a0peptide\t\r \u00a0fractionation\t\r \u00a0using\t\r \u00a0ampholytes\t\r \u00a0within\t\r \u00a0a\t\r \u00a0pH\t\r \u00a0range\t\r \u00a0from\t\r \u00a03\t\r \u00a0to\t\r \u00a010.\t\r \u00a0The\t\r \u00a0solid\t\r \u00a0curve\t\r \u00a0represents\t\r \u00a0the\t\r \u00a0average\t\r \u00a0of\t\r \u00a0three\t\r \u00a0replicates\t\r \u00a0of\t\r \u00a0peptide\t\r \u00a0fractionation\t\r \u00a0using\t\r \u00a0ampholytes\t\r \u00a0within\t\r \u00a0a\t\r \u00a0pH\t\r \u00a0range\t\r \u00a0from\t\r \u00a03\t\r \u00a0to\t\r \u00a06.\t\r \u00a0Error\t\r \u00a0bars\t\r \u00a0shown\t\r \u00a0on\t\r \u00a0the\t\r \u00a0blue\t\r \u00a0curve\t\r \u00a0represent\t\r \u00a0on\t\r \u00a0standard\t\r \u00a0deviation.\t\r \u00a0\t\r \u00a0 3.3.3\t\r \u00a0Binding\t\r \u00a0capacity\t\r \u00a0of\t\r \u00a0TiO2\t\r \u00a0for\t\r \u00a0phosphopeptides\t\r \u00a0It\t\r \u00a0is\t\r \u00a0important\t\r \u00a0to\t\r \u00a0know\t\r \u00a0the\t\r \u00a0binding\t\r \u00a0capacity\t\r \u00a0of\t\r \u00a0any\t\r \u00a0chromatographic\t\r \u00a0resin\t\r \u00a0before\t\r \u00a0loading\t\r \u00a0a\t\r \u00a0sample\t\r \u00a0so\t\r \u00a0that\t\r \u00a0the\t\r \u00a0resin\t\r \u00a0is\t\r \u00a0not\t\r \u00a0over\t\r \u00a0or\t\r \u00a0underloaded,\t\r \u00a0which,\t\r \u00a0in\t\r \u00a0the\t\r \u00a0case\t\r \u00a0of\t\r \u00a0TiO2,\t\r \u00a0could\t\r \u00a0lead\t\r \u00a0to\t\r \u00a0biased\t\r \u00a0retention\t\r \u00a0of\t\r \u00a0specific\t\r \u00a0phosphopeptides,\t\r \u00a0needless\t\r \u00a0waste\t\r \u00a0of\t\r \u00a0costly\t\r \u00a0TiO2\t\r \u00a0resin\t\r \u00a0or\t\r \u00a0undesired\t\r \u00a0retention\t\r \u00a0of\t\r \u00a0non-\u00ad\u2010phosphopeptides.\t\r \u00a0By\t\r \u00a0holding\t\r \u00a0steady\t\r \u00a0the\t\r \u00a0amount\t\r \u00a0of\t\r \u00a0TiO2\t\r \u00a0used\t\r \u00a0and\t\r \u00a0varying\t\r \u00a0the\t\r \u00a0total\t\r \u00a0digested\t\r \u00a0protein\t\r \u00a0mass\t\r \u00a0loaded\t\r \u00a0on-\u00ad\u2010column,\t\r \u00a0we\t\r \u00a0observed\t\r \u00a0an\t\r \u00a0optimal\t\r \u00a0TiO2:protein\t\r \u00a0mass\t\r \u00a0ratio\t\r \u00a0of\t\r \u00a0approximately\t\r \u00a06:1\t\r \u00a0(Figure\t\r \u00a03.3A).\t\r \u00a0We\t\r \u00a0also\t\r \u00a0observed\t\r \u00a0that\t\r \u00a0when\t\r \u00a0the\t\r \u00a0amount\t\r \u00a0of\t\r \u00a0protein\t\r \u00a0 \t\r \u00a0\t\r \u00a0 101\t\r \u00a0 loaded\t\r \u00a0was\t\r \u00a0approximately\t\r \u00a0equal\t\r \u00a0to\t\r \u00a0the\t\r \u00a0binding\t\r \u00a0capacity\t\r \u00a0of\t\r \u00a0TiO2\t\r \u00a0(6:1),\t\r \u00a0the\t\r \u00a0yield\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0was\t\r \u00a095+%\t\r \u00a0(Figure\t\r \u00a03.3B).\t\r \u00a0\t\r \u00a0 Figure\t\r \u00a03.3\t\r \u00a0Phosphopeptide\t\r \u00a0enrichment\t\r \u00a0by\t\r \u00a0MOC\t\r \u00a0 \t\r \u00a0A)\t\r \u00a0Capacity\t\r \u00a0of\t\r \u00a0titanium\t\r \u00a0dioxide\t\r \u00a0matrix\t\r \u00a0for\t\r \u00a0phosphopeptides.\t\r \u00a0Equal\t\r \u00a0masses\t\r \u00a0of\t\r \u00a0Titansphere\t\r \u00a0beads\t\r \u00a0(10\t\r \u00a0\u03bcm\t\r \u00a0diameter)\t\r \u00a0were\t\r \u00a0titrated\t\r \u00a0with\t\r \u00a0increasing\t\r \u00a0protein\t\r \u00a0amounts\t\r \u00a0for\t\r \u00a0phosphopeptide\t\r \u00a0enrichment.\t\r \u00a0Three\t\r \u00a0replicates\t\r \u00a0of\t\r \u00a0each\t\r \u00a0protein\t\r \u00a0amount\t\r \u00a0were\t\r \u00a0averaged.\t\r \u00a0Error\t\r \u00a0bars\t\r \u00a0represent\t\r \u00a0one\t\r \u00a0standard\t\r \u00a0deviation.\t\r \u00a0B)\t\r \u00a0The\t\r \u00a0percent\t\r \u00a0yield\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0for\t\r \u00a0each\t\r \u00a0condition\t\r \u00a0in\t\r \u00a0A\t\r \u00a0is\t\r \u00a0shown.\t\r \u00a0\t\r \u00a0 3.3.4\t\r \u00a0Analysis\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0by\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0One\t\r \u00a0property\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0that\t\r \u00a0has\t\r \u00a0been\t\r \u00a0assumed\t\r \u00a0to\t\r \u00a0limit\t\r \u00a0phosphoproteomics\t\r \u00a0is\t\r \u00a0the\t\r \u00a0relative\t\r \u00a0hydrophilicity\t\r \u00a0imparted\t\r \u00a0by\t\r \u00a0the\t\r \u00a0phosphate\t\r \u00a0group,\t\r \u00a0 \t\r \u00a0\t\r \u00a0 102\t\r \u00a0 which\t\r \u00a0could\t\r \u00a0potentially\t\r \u00a0require\t\r \u00a0altered\t\r \u00a0chromatographic\t\r \u00a0gradients\t\r \u00a0or\t\r \u00a0even\t\r \u00a0different\t\r \u00a0phases.\t\r \u00a0In\t\r \u00a0the\t\r \u00a0process\t\r \u00a0of\t\r \u00a0optimizing\t\r \u00a0phosphopeptide\t\r \u00a0analysis,\t\r \u00a0we\t\r \u00a0tested\t\r \u00a0several\t\r \u00a0gradient\t\r \u00a0profiles\t\r \u00a0but\t\r \u00a0found\t\r \u00a0that\t\r \u00a0our\t\r \u00a0standard\t\r \u00a0profile\t\r \u00a0for\t\r \u00a0conventional\t\r \u00a0LC-\u00ad\u2010MSn,\t\r \u00a0which\t\r \u00a0spends\t\r \u00a0the\t\r \u00a0majority\t\r \u00a0of\t\r \u00a0analysis\t\r \u00a0time\t\r \u00a0between\t\r \u00a03%\t\r \u00a0and\t\r \u00a030%\t\r \u00a0acetonitrile,\t\r \u00a0was\t\r \u00a0as\t\r \u00a0good\t\r \u00a0as\t\r \u00a0any\t\r \u00a0other,\t\r \u00a0with\t\r \u00a0an\t\r \u00a0excellent\t\r \u00a0distribution\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0across\t\r \u00a0the\t\r \u00a0timescale\t\r \u00a0(Figure\t\r \u00a03.4).\t\r \u00a0 Figure\t\r \u00a03.4\t\r \u00a0Elution\t\r \u00a0times\t\r \u00a0of\t\r \u00a0phosphorylated\t\r \u00a0versus\t\r \u00a0non-\u00ad\u2010phosphorylated\t\r \u00a0peptides\t\r \u00a0from\t\r \u00a0a\t\r \u00a0C18\t\r \u00a0 reversed\t\r \u00a0phase\t\r \u00a0column\t\r \u00a0 Elution\t\r \u00a0time\t\r \u00a0versus\t\r \u00a0m\/z\t\r \u00a0of\t\r \u00a0all\t\r \u00a0peptides\t\r \u00a0detected\t\r \u00a0in\t\r \u00a0a\t\r \u00a0sample\t\r \u00a0enriched\t\r \u00a0for\t\r \u00a0phosphopeptides\t\r \u00a0is\t\r \u00a0overlaid\t\r \u00a0over\t\r \u00a0top\t\r \u00a0of\t\r \u00a0the\t\r \u00a0total\t\r \u00a0ion\t\r \u00a0chromatogram.\t\r \u00a0Phosphopeptides\t\r \u00a0are\t\r \u00a0shown\t\r \u00a0in\t\r \u00a0red\t\r \u00a0and\t\r \u00a0non-\u00ad\u2010phosphopeptides\t\r \u00a0are\t\r \u00a0shown\t\r \u00a0in\t\r \u00a0blue.\t\r \u00a0\t\r \u00a0\t\r \u00a0 The\t\r \u00a0solvents\t\r \u00a0used\t\r \u00a0in\t\r \u00a0most\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0applications\t\r \u00a0designed\t\r \u00a0to\t\r \u00a0analyze\t\r \u00a0peptides\t\r \u00a0have\t\r \u00a0a\t\r \u00a0pH\t\r \u00a0of\t\r \u00a0between\t\r \u00a02.5\t\r \u00a0and\t\r \u00a03.0,\t\r \u00a0e.g.,\t\r \u00a0pH\t\r \u00a0of\t\r \u00a00.5%\t\r \u00a0acetic\t\r \u00a0acid\t\r \u00a0=\t\r \u00a02.9,\t\r \u00a0pH\t\r \u00a0of\t\r \u00a00.1%\t\r \u00a0formic\t\r \u00a0acid\t\r \u00a0=\t\r \u00a02.7.\t\r \u00a0However,\t\r \u00a0the\t\r \u00a0pKa\t\r \u00a0of\t\r \u00a0the\t\r \u00a0first\t\r \u00a0proton\t\r \u00a0from\t\r \u00a0phosphoric\t\r \u00a0acid\t\r \u00a0is\t\r \u00a02.15\t\r \u00a0and\t\r \u00a0likely\t\r \u00a0even\t\r \u00a0lower\t\r \u00a0within\t\r \u00a0the\t\r \u00a0physiochemical\t\r \u00a0environment\t\r \u00a0of\t\r \u00a0a\t\r \u00a0peptide,\t\r \u00a0meaning\t\r \u00a0that\t\r \u00a0phosphates\t\r \u00a0will\t\r \u00a0typically\t\r \u00a0carry\t\r \u00a0a\t\r \u00a0negative\t\r \u00a0charge\t\r \u00a0under\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0conditions.\t\r \u00a0In\t\r \u00a0data-\u00ad\u2010dependent\t\r \u00a0MSn\t\r \u00a0mode\t\r \u00a0for\t\r \u00a0peptide\t\r \u00a0analyses,\t\r \u00a0mass\t\r \u00a0spectrometers\t\r \u00a0are\t\r \u00a0typically\t\r \u00a0directed\t\r \u00a0to\t\r \u00a0only\t\r \u00a0select\t\r \u00a0multiply-\u00ad\u2010charged\t\r \u00a0precursor\t\r \u00a0ions\t\r \u00a0for\t\r \u00a0fragmentation.\t\r \u00a0However\t\r \u00a0when\t\r \u00a0we\t\r \u00a0allowed\t\r \u00a0singly-\u00ad\u2010charged\t\r \u00a0ions\t\r \u00a0to\t\r \u00a0be\t\r \u00a0sequenced\t\r \u00a0as\t\r \u00a0well,\t\r \u00a0we\t\r \u00a0observed\t\r \u00a0 \t\r \u00a0\t\r \u00a0 103\t\r \u00a0 essentially\t\r \u00a0no\t\r \u00a0difference\t\r \u00a0in\t\r \u00a0either\t\r \u00a0the\t\r \u00a0number\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0identified\t\r \u00a0or\t\r \u00a0the\t\r \u00a0numbers\t\r \u00a0of\t\r \u00a0phosphates\t\r \u00a0per\t\r \u00a0peptide\t\r \u00a0(Figure\t\r \u00a03.5A,\t\r \u00a0B).\t\r \u00a0\t\r \u00a0Historically,\t\r \u00a0there\t\r \u00a0has\t\r \u00a0been\t\r \u00a0a\t\r \u00a0perception\t\r \u00a0in\t\r \u00a0the\t\r \u00a0mass\t\r \u00a0spectrometry\t\r \u00a0field\t\r \u00a0that\t\r \u00a0phosphopeptides\t\r \u00a0will\t\r \u00a0fragment\t\r \u00a0poorly\t\r \u00a0in\t\r \u00a0positive\t\r \u00a0ion\t\r \u00a0mode,\t\r \u00a0with\t\r \u00a0neutral\t\r \u00a0loss\t\r \u00a0of\t\r \u00a0phosphoric\t\r \u00a0acid\t\r \u00a0(H3PO4,\t\r \u00a0from\t\r \u00a0pS\t\r \u00a0and\t\r \u00a0pT)\t\r \u00a0often\t\r \u00a0being\t\r \u00a0preferred\t\r \u00a0to\t\r \u00a0backbone\t\r \u00a0cleavage(489).\t\r \u00a0We\t\r \u00a0therefore\t\r \u00a0tested\t\r \u00a0the\t\r \u00a0utility\t\r \u00a0of\t\r \u00a0the\t\r \u00a0MultiStage\t\r \u00a0activation\t\r \u00a0fragmentation\t\r \u00a0mechanism\t\r \u00a0available\t\r \u00a0on\t\r \u00a0the\t\r \u00a0LTQ-\u00ad\u2010Orbitrap\t\r \u00a0for\t\r \u00a0phosphopeptide\t\r \u00a0analysis.\t\r \u00a0As\t\r \u00a0reported\t\r \u00a0by\t\r \u00a0others(422),\t\r \u00a0the\t\r \u00a0additional\t\r \u00a0time\t\r \u00a0required\t\r \u00a0for\t\r \u00a0multi-\u00ad\u2010stage\t\r \u00a0activation\t\r \u00a0experiments\t\r \u00a0seems\t\r \u00a0to\t\r \u00a0have\t\r \u00a0a\t\r \u00a0small\t\r \u00a0negative\t\r \u00a0impact\t\r \u00a0on\t\r \u00a0the\t\r \u00a0overall\t\r \u00a0number\t\r \u00a0of\t\r \u00a0phosphopeptide\t\r \u00a0identifications\t\r \u00a0(Figure\t\r \u00a03.5C)\t\r \u00a0and\t\r \u00a0it\t\r \u00a0also\t\r \u00a0does\t\r \u00a0not\t\r \u00a0appear\t\r \u00a0to\t\r \u00a0improve\t\r \u00a0traditional\t\r \u00a0MS2\t\r \u00a0with\t\r \u00a0regards\t\r \u00a0to\t\r \u00a0the\t\r \u00a0number\t\r \u00a0of\t\r \u00a0multiply-\u00ad\u2010phosphorylated\t\r \u00a0peptides\t\r \u00a0identified\t\r \u00a0(Figure\t\r \u00a03.5D).\t\r \u00a0Another\t\r \u00a0potential\t\r \u00a0advantage\t\r \u00a0of\t\r \u00a0MS3\t\r \u00a0or\t\r \u00a0pseudo-\u00ad\u2010MS3\t\r \u00a0methods\t\r \u00a0is\t\r \u00a0in\t\r \u00a0providing\t\r \u00a0more\t\r \u00a0backbone\t\r \u00a0cleavage,\t\r \u00a0leading\t\r \u00a0to\t\r \u00a0more\t\r \u00a0MSn\t\r \u00a0ions\t\r \u00a0available\t\r \u00a0for\t\r \u00a0peptide\t\r \u00a0identification.\t\r \u00a0However,\t\r \u00a0while\t\r \u00a0there\t\r \u00a0appeared\t\r \u00a0to\t\r \u00a0be\t\r \u00a0a\t\r \u00a0very\t\r \u00a0small\t\r \u00a0shift\t\r \u00a0towards\t\r \u00a0higher\t\r \u00a0Mascot\t\r \u00a0IonsScores\t\r \u00a0when\t\r \u00a0using\t\r \u00a0MultiStage\t\r \u00a0activation,\t\r \u00a0the\t\r \u00a0differences\t\r \u00a0were\t\r \u00a0well\t\r \u00a0within\t\r \u00a0the\t\r \u00a0margin\t\r \u00a0of\t\r \u00a0error\t\r \u00a0of\t\r \u00a0the\t\r \u00a0experiments,\t\r \u00a0both\t\r \u00a0for\t\r \u00a0the\t\r \u00a0initial\t\r \u00a0IonsScore\t\r \u00a0and\t\r \u00a0the\t\r \u00a0subsequent\t\r \u00a0PTM\t\r \u00a0Score(496)(Figure\t\r \u00a03.5E,\t\r \u00a0F).\t\r \u00a0\t\r \u00a0 \t\r \u00a0\t\r \u00a0 104\t\r \u00a0 Figure\t\r \u00a03.5\t\r \u00a0Analyzing\t\r \u00a0phosphopeptides\t\r \u00a0by\t\r \u00a0LC-\u00ad\u2010ESI-\u00ad\u2010MSn\t\r \u00a0 \t\r \u00a0\t\r \u00a0A)\t\r \u00a0Number\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0identified\t\r \u00a0by\t\r \u00a0including\t\r \u00a0versus\t\r \u00a0excluding\t\r \u00a0singly\t\r \u00a0charged\t\r \u00a0peptides\t\r \u00a0for\t\r \u00a0MSn\t\r \u00a0B)\t\r \u00a0Distribution\t\r \u00a0of\t\r \u00a0multiply\t\r \u00a0phosphorylated\t\r \u00a0peptides\t\r \u00a0identified\t\r \u00a0by\t\r \u00a0including\t\r \u00a0versus\t\r \u00a0excluding\t\r \u00a0singly\t\r \u00a0charged\t\r \u00a0peptides\t\r \u00a0for\t\r \u00a0MSn.\t\r \u00a0C)\t\r \u00a0Number\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0identified\t\r \u00a0using\t\r \u00a0MS2\t\r \u00a0versus\t\r \u00a0MultiStage\t\r \u00a0Activation.\t\r \u00a0D)\t\r \u00a0Distribution\t\r \u00a0of\t\r \u00a0multiply\t\r \u00a0phosphorylated\t\r \u00a0peptides\t\r \u00a0identified\t\r \u00a0using\t\r \u00a0MS2\t\r \u00a0versus\t\r \u00a0MultiStage\t\r \u00a0Activation.\t\r \u00a0E)\t\r \u00a0Distribution\t\r \u00a0of\t\r \u00a0Mascot\t\r \u00a0scores\t\r \u00a0using\t\r \u00a0MS2\t\r \u00a0versus\t\r \u00a0MultiStage\t\r \u00a0Activation.\t\r \u00a0F)\t\r \u00a0Distribution\t\r \u00a0of\t\r \u00a0PTM\t\r \u00a0scores\t\r \u00a0from\t\r \u00a0MSQuant\t\r \u00a0using\t\r \u00a0MS2\t\r \u00a0versus\t\r \u00a0MultiStage\t\r \u00a0Activation.\t\r \u00a0Data\t\r \u00a0in\t\r \u00a0A\t\r \u00a0and\t\r \u00a0C\t\r \u00a0represent\t\r \u00a0results\t\r \u00a0from\t\r \u00a0two\t\r \u00a0independent\t\r \u00a0experiments\t\r \u00a0that\t\r \u00a0started\t\r \u00a0with\t\r \u00a01500\t\r \u00a0or\t\r \u00a0500\t\r \u00a0\u03bcg\t\r \u00a0of\t\r 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\u00a0pre-\u00ad\u2010treating\t\r \u00a0cells\t\r \u00a0with\t\r \u00a0calyculin\t\r \u00a0A,\t\r \u00a0sodium\t\r \u00a0pervanadate,\t\r \u00a0or\t\r \u00a0two\t\r \u00a0phosphatase\t\r \u00a0ihibitor\t\r \u00a0cocktails\t\r \u00a0resulted\t\r \u00a0in\t\r \u00a0only\t\r \u00a0a\t\r \u00a010-\u00ad\u201040%\t\r \u00a0increase\t\r \u00a0in\t\r \u00a0the\t\r \u00a0number\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0identified\t\r \u00a0by\t\r \u00a0LC-\u00ad\u2010MSn(388).\t\r \u00a0In\t\r \u00a0a\t\r \u00a0similar\t\r \u00a0study\t\r \u00a0using\t\r \u00a0pervanatate,\t\r \u00a0calyculin\t\r \u00a0A\t\r \u00a0and\t\r \u00a0deltamethrin,\t\r \u00a0Pan\t\r \u00a0et\t\r \u00a0al.\t\r \u00a0found\t\r \u00a0only\t\r \u00a027%\t\r \u00a0of\t\r \u00a0all\t\r \u00a0phosphopeptides\t\r \u00a0to\t\r \u00a0increase\t\r \u00a0more\t\r \u00a0than\t\r \u00a0two\t\r \u00a0fold(387).\t\r \u00a0In\t\r \u00a0both\t\r \u00a0cases\t\r \u00a0the\t\r \u00a0authors\t\r \u00a0report\t\r \u00a0that\t\r 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\u00a0free\t\r \u00a0phosphate\t\r \u00a0still\t\r \u00a0detected\t\r \u00a0in\t\r \u00a0deoxycholate\/boiled\t\r \u00a0samples\t\r \u00a0(Figure\t\r \u00a03.1A)\t\r \u00a0likely\t\r \u00a0reflect\t\r \u00a0phosphate\t\r \u00a0that\t\r \u00a0was\t\r \u00a0not\t\r \u00a0directly\t\r \u00a0liberated\t\r \u00a0from\t\r \u00a0protein\t\r \u00a0substrates\t\r \u00a0(i.e.,\t\r \u00a0from\t\r \u00a0inositol\t\r \u00a0phosphatases\t\r \u00a0or\t\r \u00a0from\t\r \u00a0ATP).\t\r \u00a0A\t\r \u00a0harsh\t\r \u00a0denatuation\t\r \u00a0method\t\r \u00a0also\t\r \u00a0helps\t\r \u00a0to\t\r \u00a0avoid\t\r \u00a0downstream\t\r \u00a0complications\t\r \u00a0caused\t\r \u00a0by\t\r \u00a0inhibitors\t\r \u00a0during\t\r \u00a0phosphopeptide\t\r \u00a0enrichment.\t\r \u00a0The\t\r \u00a0vast\t\r \u00a0majority\t\r \u00a0of\t\r \u00a0phosphatase\t\r \u00a0inhibitors\t\r \u00a0are\t\r \u00a0designed\t\r \u00a0to\t\r \u00a0mimic\t\r \u00a0phosphate\t\r \u00a0and\t\r \u00a0bind\t\r \u00a0the\t\r \u00a0enzymes\u2019\t\r \u00a0active\t\r \u00a0sites.\t\r \u00a0Thus,\t\r \u00a0several\t\r \u00a0inhibitors\t\r \u00a0contain\t\r \u00a0phosphate\t\r \u00a0groups\t\r \u00a0and\t\r \u00a0likely\t\r \u00a0have\t\r \u00a0a\t\r \u00a0high\t\r \u00a0affinity\t\r \u00a0for\t\r \u00a0TiO2.\t\r \u00a0While\t\r \u00a0testing\t\r \u00a0a\t\r \u00a0cocktail\t\r \u00a0of\t\r \u00a0phosphatase\t\r \u00a0inhibitors\t\r \u00a0present\t\r \u00a0during\t\r \u00a0phosphopeptide\t\r \u00a0enrichment,\t\r \u00a0pyrophosphate,\t\r \u00a0\u03b2-\u00ad\u2010glycerophosphate\t\r \u00a0and\t\r \u00a0calyculin\t\r \u00a0A\t\r \u00a0all\t\r \u00a0resulted\t\r \u00a0in\t\r \u00a0between\t\r \u00a0a\t\r \u00a0three\t\r \u00a0and\t\r \u00a0five\t\r \u00a0fold\t\r \u00a0decrease\t\r \u00a0in\t\r \u00a0the\t\r \u00a0number\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0identified\t\r \u00a0by\t\r \u00a0LC-\u00ad\u2010MSn.\t\r \u00a0Thus,\t\r \u00a0for\t\r \u00a0efficient\t\r \u00a0phosphopeptide\t\r \u00a0recovery,\t\r \u00a0either\t\r \u00a0an\t\r \u00a0excess\t\r \u00a0of\t\r \u00a0costly\t\r \u00a0matrix\t\r \u00a0must\t\r \u00a0be\t\r \u00a0used,\t\r \u00a0or\t\r \u00a0phosphatase\t\r \u00a0inhibitors\t\r \u00a0should\t\r \u00a0be\t\r \u00a0depleted\t\r \u00a0(i.e.\t\r \u00a0using\t\r \u00a0chromatographic\t\r \u00a0or\t\r \u00a0protein\t\r \u00a0precipitation\t\r \u00a0methods)\t\r \u00a0prior\t\r \u00a0to\t\r \u00a0 \t\r \u00a0\t\r \u00a0 106\t\r \u00a0 enrichment.\t\r \u00a0However\t\r \u00a0this\t\r \u00a0requires\t\r \u00a0additional\t\r \u00a0unnecessary\t\r \u00a0sample\t\r \u00a0handling\t\r \u00a0steps,\t\r \u00a0while\t\r \u00a0samples\t\r \u00a0solubilized\t\r \u00a0in\t\r \u00a0deoxycholate\t\r \u00a0can\t\r \u00a0be\t\r \u00a0directly\t\r \u00a0used\t\r \u00a0for\t\r \u00a0both\t\r \u00a0trypsinization\t\r \u00a0and\t\r \u00a0phosphopeptide\t\r \u00a0enrichment.\t\r \u00a0We\t\r \u00a0did\t\r \u00a0not\t\r \u00a0test\t\r \u00a0other\t\r \u00a0chaotropes\t\r \u00a0such\t\r \u00a0as\t\r \u00a0high\t\r \u00a0concentrations\t\r \u00a0of\t\r \u00a0urea\t\r \u00a0in\t\r \u00a0this\t\r \u00a0study,\t\r \u00a0as\t\r \u00a0many\t\r \u00a0enzymes\t\r \u00a0retain\t\r \u00a0their\t\r \u00a0activity\t\r \u00a0at\t\r \u00a0RT\t\r \u00a0in\t\r \u00a0these\t\r \u00a0conditions,\t\r \u00a0e.g.,\t\r \u00a0endopeptidase\t\r \u00a0LysC(467)\t\r \u00a0and\t\r \u00a0heating\t\r \u00a0urea\t\r \u00a0solutions\t\r \u00a0can\t\r \u00a0result\t\r \u00a0in\t\r \u00a0significant,\t\r \u00a0undesireable\t\r \u00a0carbamylation(497,\t\r \u00a0498).\t\r \u00a0The\t\r \u00a0dynamic\t\r \u00a0range\t\r \u00a0of\t\r \u00a0a\t\r \u00a0mammalian\t\r \u00a0cell\u2019s\t\r \u00a0proteome\t\r \u00a0can\t\r \u00a0span\t\r \u00a0over\t\r \u00a0ten\t\r \u00a0orders\t\r \u00a0of\t\r \u00a0magnitude,\t\r \u00a0while\t\r \u00a0current\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0systems\t\r \u00a0can\t\r \u00a0handle\t\r \u00a0only\t\r \u00a0four\t\r \u00a0at\t\r \u00a0best(499,\t\r \u00a0500).\t\r \u00a0Thus,\t\r \u00a0the\t\r \u00a0need\t\r \u00a0for\t\r \u00a0sample\t\r \u00a0pre-\u00ad\u2010fractionation\t\r \u00a0is\t\r \u00a0widely\t\r \u00a0acknowledged\t\r \u00a0by\t\r \u00a0researchers\t\r \u00a0in\t\r \u00a0proteomics.\t\r \u00a0Here\t\r \u00a0we\t\r \u00a0use\t\r \u00a0in-\u00ad\u2010solution\t\r \u00a0IEF\t\r \u00a0with\t\r \u00a0a\t\r \u00a0loading\t\r \u00a0capacity\t\r \u00a0of\t\r \u00a030\t\r \u00a0mg\t\r \u00a0to\t\r \u00a0fractionate\t\r \u00a0phosphopeptides\t\r \u00a0effectively\t\r \u00a0and\t\r \u00a0uniformly\t\r \u00a0across\t\r \u00a0a\t\r \u00a0pH\t\r \u00a0range\t\r \u00a0of\t\r \u00a03\t\r \u00a0to\t\r \u00a06\t\r \u00a0within\t\r \u00a0a\t\r \u00a0few\t\r \u00a0hours.\t\r \u00a0An\t\r \u00a0additional\t\r \u00a0advantage\t\r \u00a0of\t\r \u00a0solution-\u00ad\u2010based\t\r \u00a0IEF\t\r \u00a0is\t\r \u00a0the\t\r \u00a0virtually\t\r \u00a0complete\t\r \u00a0sample\t\r \u00a0recovery,\t\r \u00a0in\t\r \u00a0contrast\t\r \u00a0to\t\r \u00a0immobilized\t\r \u00a0pH\t\r \u00a0gradient\t\r \u00a0(IPG)-\u00ad\u2010based\t\r \u00a0IEF\t\r \u00a0and\t\r \u00a0several\t\r \u00a0chromatographic\t\r \u00a0systems\t\r \u00a0where\t\r \u00a0peptides\t\r \u00a0with\t\r \u00a0extreme\t\r \u00a0pIs,\t\r \u00a0charge\t\r \u00a0states(364),\t\r \u00a0etc.\t\r \u00a0migrate\t\r \u00a0off\t\r \u00a0the\t\r \u00a0strip,\t\r \u00a0are\t\r \u00a0not\t\r \u00a0retained\t\r \u00a0on-\u00ad\u2010column\t\r \u00a0or\t\r \u00a0bind\t\r \u00a0irreversibly\t\r \u00a0to\t\r \u00a0the\t\r \u00a0resin.\t\r \u00a0Solution-\u00ad\u2010based\t\r \u00a0IEF\t\r \u00a0is\t\r \u00a0also\t\r \u00a0considerably\t\r \u00a0faster\t\r \u00a0to\t\r \u00a0perform\t\r \u00a0than\t\r \u00a0IPG-\u00ad\u2010based\t\r \u00a0IEF,\t\r \u00a0which\t\r \u00a0can\t\r \u00a0require\t\r \u00a0run\t\r \u00a0times\t\r \u00a0of\t\r \u00a020\t\r \u00a0to\t\r \u00a024\t\r \u00a0h(362).\t\r \u00a0Two\t\r \u00a0previous\t\r \u00a0reports\t\r \u00a0evaluating\t\r \u00a0MS3\t\r \u00a0and\t\r \u00a0pseudo-\u00ad\u2010MS3\t\r \u00a0for\t\r \u00a0phosphopeptide\t\r \u00a0analysis\t\r \u00a0both\t\r \u00a0agree\t\r \u00a0that\t\r \u00a0MS3\t\r \u00a0methods\t\r \u00a0perform\t\r \u00a0poorly\t\r \u00a0compared\t\r \u00a0to\t\r \u00a0the\t\r \u00a0others,\t\r \u00a0However,\t\r \u00a0Villen\t\r \u00a0et\t\r \u00a0al.\t\r \u00a0found\t\r \u00a0that\t\r \u00a0MultiStage\t\r \u00a0Activation\t\r \u00a0outperforms\t\r \u00a0MS2\t\r \u00a0while\t\r \u00a0Ulintz\t\r \u00a0 et\t\r \u00a0al.\t\r \u00a0found\t\r \u00a0exactly\t\r \u00a0the\t\r \u00a0opposite.\t\r \u00a0As\t\r \u00a0our\t\r \u00a0data\t\r \u00a0clearly\t\r \u00a0favour\t\r \u00a0MS2\t\r \u00a0over\t\r \u00a0MultiStage\t\r \u00a0Activation,\t\r \u00a0we\t\r \u00a0feel\t\r \u00a0that\t\r \u00a0the\t\r \u00a0balance\t\r \u00a0of\t\r \u00a0evidence\t\r \u00a0now\t\r \u00a0suggests\t\r \u00a0that\t\r \u00a0conventional\t\r \u00a0MS2\t\r \u00a0of\t\r \u00a0precursors\t\r \u00a0with\t\r \u00a0two\t\r \u00a0or\t\r \u00a0more\t\r \u00a0charges\t\r \u00a0out-\u00ad\u2010performs\t\r \u00a0MS3\t\r \u00a0and\t\r \u00a0pseudo-\u00ad\u2010MS3\t\r \u00a0 \t\r \u00a0\t\r \u00a0 107\t\r \u00a0 approaches(422,\t\r \u00a0423).\t\r \u00a0This\t\r \u00a0is\t\r \u00a0likely\t\r \u00a0due\t\r \u00a0to\t\r \u00a0the\t\r \u00a0fact\t\r \u00a0that\t\r \u00a0higher-\u00ad\u2010order\t\r \u00a0scans\t\r \u00a0are\t\r \u00a0slower\t\r \u00a0and\t\r \u00a0generate\t\r \u00a0ion\t\r \u00a0fragments\t\r \u00a0with\t\r \u00a0lower\t\r \u00a0intensities\t\r \u00a0than\t\r \u00a0those\t\r \u00a0generated\t\r \u00a0by\t\r \u00a0an\t\r \u00a0MS2\t\r \u00a0scan(422).\t\r \u00a0The\t\r \u00a0previous\t\r \u00a0concerns\t\r \u00a0that\t\r \u00a0the\t\r \u00a0negative\t\r \u00a0charges\t\r \u00a0on\t\r \u00a0phosphates\t\r \u00a0would\t\r \u00a0cancel\t\r \u00a0out\t\r \u00a0the\t\r \u00a0positive\t\r \u00a0charges\t\r \u00a0on\t\r \u00a0the\t\r \u00a0rest\t\r \u00a0of\t\r \u00a0a\t\r \u00a0peptide\t\r \u00a0molecule\t\r \u00a0are\t\r \u00a0unfounded\t\r \u00a0as\t\r \u00a0charge\t\r \u00a0state\t\r \u00a0in\t\r \u00a0the\t\r \u00a0gas\t\r \u00a0phase\t\r \u00a0is\t\r \u00a0clearly\t\r \u00a0not\t\r \u00a0equivalent\t\r \u00a0to\t\r \u00a0solution\t\r \u00a0charge\t\r \u00a0state\t\r \u00a0calculated\t\r \u00a0from\t\r \u00a0the\t\r \u00a0acid\t\r \u00a0dissociation\t\r \u00a0constants\t\r \u00a0of\t\r \u00a0all\t\r \u00a0ionizable\t\r \u00a0groups.\t\r \u00a0Undoubtedly,\t\r \u00a0for\t\r \u00a0complete\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0a\t\r \u00a0phosphoproteome\t\r \u00a0where\t\r \u00a0every\t\r \u00a0single\t\r \u00a0phosphopeptide\t\r \u00a0is\t\r \u00a0fragmented\t\r \u00a0and\t\r \u00a0quantified,\t\r \u00a0a\t\r \u00a0combination\t\r \u00a0of\t\r \u00a0methods\t\r \u00a0will\t\r \u00a0be\t\r \u00a0required,\t\r \u00a0including\t\r \u00a0ETD(501,\t\r \u00a0502),\t\r \u00a0MS2,\t\r \u00a0MS3\t\r \u00a0and\t\r \u00a0even\t\r \u00a0some\t\r \u00a0of\t\r \u00a0the\t\r \u00a0methods\t\r \u00a0that\t\r \u00a0are\t\r \u00a0not\t\r \u00a0as\t\r \u00a0compatible\t\r \u00a0with\t\r \u00a0LC\t\r \u00a0timescales,\t\r \u00a0e.g.,\t\r \u00a0ECD\t\r \u00a0and\t\r \u00a0infrared\t\r \u00a0multiphoton\t\r \u00a0dissociation.\t\r \u00a0In\t\r \u00a0addition,\t\r \u00a0the\t\r \u00a0potential\t\r \u00a0problems\t\r \u00a0of\t\r \u00a0phosphosite\t\r \u00a0scrambling\t\r \u00a0in\t\r \u00a0ion\t\r \u00a0trap\t\r \u00a0fragmentation\t\r \u00a0requires\t\r \u00a0significant\t\r \u00a0work\t\r \u00a0in\t\r \u00a0order\t\r \u00a0to\t\r \u00a0allow\t\r \u00a0accurate assignment\t\r \u00a0of\t\r \u00a0the\t\r \u00a0specific\t\r \u00a0residues\t\r \u00a0that\t\r \u00a0are\t\r \u00a0modified(425).\t\r \u00a0Several\t\r \u00a0factors\t\r \u00a0still\t\r \u00a0limit\t\r \u00a0our\t\r \u00a0ability\t\r \u00a0for\t\r \u00a0a\t\r \u00a0truly\t\r \u00a0complete\t\r \u00a0phosphoproteomic\t\r \u00a0study(489),\t\r \u00a0but\t\r \u00a0while\t\r \u00a0those\t\r \u00a0are\t\r \u00a0still\t\r \u00a0being\t\r \u00a0addressed\t\r \u00a0the\t\r \u00a0methods\t\r \u00a0described\t\r \u00a0here\t\r \u00a0can\t\r \u00a0be\t\r \u00a0implemented\t\r \u00a0immediately\t\r \u00a0and\t\r \u00a0applied\t\r \u00a0to\t\r \u00a0answer\t\r \u00a0an\t\r \u00a0array\t\r \u00a0of\t\r \u00a0biological\t\r \u00a0questions. \t\r \u00a0\t\r \u00a0 108\t\r \u00a0 4\t\r \u00a0SopB\t\r \u00a0is\t\r \u00a0a\t\r \u00a0master\t\r \u00a0regulator\t\r \u00a0of\t\r \u00a0host\t\r \u00a0signaling\t\r \u00a0cascades\t\r \u00a0 during\t\r \u00a0Salmonella\t\r \u00a0infection\t\r \u00a0 4.1\t\r \u00a0Introduction\t\r \u00a0 Salmonella\t\r \u00a0enterica\t\r \u00a0is\t\r \u00a0a\t\r \u00a0food-\u00ad\u2010borne,\t\r \u00a0facultative\t\r \u00a0intracellular\t\r \u00a0bacterium\t\r \u00a0that\t\r \u00a0causes\t\r \u00a0gastroenteritis\t\r \u00a0and\t\r \u00a0typhoid\t\r \u00a0fever\t\r \u00a0leading\t\r \u00a0to\t\r \u00a0significant\t\r \u00a0human\t\r \u00a0morbidity\t\r \u00a0and\t\r \u00a0mortality(452).\t\r \u00a0The\t\r \u00a0various\t\r \u00a0serovars\t\r \u00a0of\t\r \u00a0S.\t\r \u00a0enterica\t\r \u00a0have\t\r \u00a0evolved\t\r \u00a0intricate\t\r \u00a0mechanisms\t\r \u00a0for\t\r \u00a0evading\t\r \u00a0host\t\r \u00a0immunity,\t\r \u00a0whereby\t\r \u00a0the\t\r \u00a0pathogen\t\r \u00a0induces\t\r \u00a0its\t\r \u00a0own\t\r \u00a0internalization\t\r \u00a0into\t\r \u00a0a\t\r \u00a0membrane-\u00ad\u2010bound\t\r \u00a0vacuole\t\r \u00a0termed\t\r \u00a0the\t\r \u00a0SCV,\t\r \u00a0which\t\r \u00a0evades\t\r \u00a0degradation\t\r \u00a0within\t\r \u00a0host\t\r \u00a0cells(453,\t\r \u00a0503).\t\r \u00a0Effector\t\r \u00a0proteins,\t\r \u00a0translocated\t\r \u00a0into\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0by\t\r \u00a0two\t\r \u00a0T3SS\t\r \u00a0encoded\t\r \u00a0on\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0and\t\r \u00a0SPI-\u00ad\u20102,\t\r \u00a0control\t\r \u00a0the\t\r \u00a0formation\t\r \u00a0and\t\r \u00a0maturation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV(21).\t\r \u00a0While\t\r \u00a0T3SS-\u00ad\u20102\t\r \u00a0acts\t\r \u00a0at\t\r \u00a0later\t\r \u00a0stages\t\r \u00a0of\t\r \u00a0infection\t\r \u00a0to\t\r \u00a0direct\t\r \u00a0trafficking\t\r \u00a0of\t\r \u00a0and\t\r \u00a0bacterial\t\r \u00a0replication\t\r \u00a0within\t\r \u00a0the\t\r \u00a0SCV,\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0effectors\t\r \u00a0are\t\r \u00a0translocated\t\r \u00a0immediately\t\r \u00a0upon\t\r \u00a0contact\t\r \u00a0with\t\r \u00a0the\t\r \u00a0host\t\r \u00a0cell.\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0effectors\t\r \u00a0have\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0target\t\r \u00a0Rho\t\r \u00a0family\t\r \u00a0GTPases\t\r \u00a0and\t\r \u00a0actin\t\r \u00a0binding\t\r \u00a0proteins\t\r \u00a0to\t\r \u00a0induce\t\r \u00a0membrane\t\r \u00a0ruffling,\t\r \u00a0macropinocytosis,\t\r \u00a0and\t\r \u00a0nuclear\t\r \u00a0responses\t\r \u00a0leading\t\r \u00a0to\t\r \u00a0inflammation(31,\t\r \u00a0187,\t\r \u00a0189).\t\r \u00a0They\t\r \u00a0have\t\r \u00a0also\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0regulate\t\r \u00a0bacterial\t\r \u00a0replication\t\r \u00a0and\t\r \u00a0trafficking\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0host\t\r \u00a0functions\t\r \u00a0such\t\r \u00a0as\t\r \u00a0cell\t\r \u00a0death,\t\r \u00a0ion\t\r \u00a0secretion,\t\r \u00a0and\t\r \u00a0cell\t\r \u00a0signaling\t\r \u00a0through\t\r \u00a0phospholipids\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0protein\t\r \u00a0phosphorylation,\t\r \u00a0acetylation\t\r \u00a0and\t\r \u00a0ubiquitylation(57,\t\r \u00a058,\t\r \u00a0149,\t\r \u00a0164).\t\r \u00a0\t\r \u00a0Of\t\r \u00a0relevance,\t\r \u00a0SopB\t\r \u00a0(a\t\r \u00a0phosphoinositide\t\r \u00a0phosphatase)\t\r \u00a0and\t\r \u00a0SopE\/E2\t\r \u00a0(GEFs)\t\r \u00a0activate\t\r \u00a0Cdc42\t\r \u00a0to\t\r \u00a0induce\t\r \u00a0downstream\t\r \u00a0signaling\t\r \u00a0through\t\r \u00a0JNK\t\r \u00a0and\t\r \u00a0p38\t\r \u00a0isoforms,\t\r \u00a0 \t\r \u00a0\t\r \u00a0 109\t\r \u00a0 leading\t\r \u00a0to\t\r \u00a0proinflammatory\t\r \u00a0cytokine\t\r \u00a0production(45).\t\r \u00a0SopB\t\r \u00a0also\t\r \u00a0activates\t\r \u00a0Akt,\t\r \u00a0which\t\r \u00a0induces\t\r \u00a0antiapoptotic\t\r \u00a0signaling,\t\r \u00a0while\t\r \u00a0also\t\r \u00a0activating\t\r \u00a0PAK4\t\r \u00a0and\t\r \u00a0a\t\r \u00a0GAP\t\r \u00a0for\t\r \u00a0Rab14(66,\t\r \u00a0165).\t\r \u00a0Conversely,\t\r \u00a0the\t\r \u00a0effector\t\r \u00a0SptP\t\r \u00a0(GAP\t\r \u00a0and\t\r \u00a0PTP)\t\r \u00a0inactivates\t\r \u00a0Raf1\t\r \u00a0and\t\r \u00a0dephosphorylates\t\r \u00a0VCP,\t\r \u00a0while\t\r \u00a0SspH1\t\r \u00a0binds\t\r \u00a0PKN1\t\r \u00a0to\t\r \u00a0inhibit\t\r \u00a0NF-\u00ad\u2010\u03baB\t\r \u00a0dependent\t\r \u00a0gene\t\r \u00a0expression(178,\t\r \u00a0179,\t\r \u00a0183).\t\r \u00a0Finally,\t\r \u00a0AvrA\t\r \u00a0also\t\r \u00a0inhibits\t\r \u00a0MAPKs\t\r \u00a0through\t\r \u00a0acetylation,\t\r \u00a0and\t\r \u00a0prevents\t\r \u00a0NF-\u00ad\u2010\u03baB\t\r \u00a0activation\t\r \u00a0by\t\r \u00a0deubiquitylating\t\r \u00a0I-\u00ad\u2010\u03baB\u03b1(143,\t\r \u00a0144).\t\r \u00a0SipA\t\r \u00a0has\t\r \u00a0also\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0induce\t\r \u00a0PKC\t\r \u00a0activation,\t\r \u00a0leading\t\r \u00a0to\t\r \u00a0transepithalial\t\r \u00a0migration\t\r \u00a0of\t\r \u00a0PMNs(147,\t\r \u00a0192,\t\r \u00a0193).\t\r \u00a0However,\t\r \u00a0activation\t\r \u00a0of\t\r \u00a0PKC\t\r \u00a0by\t\r \u00a0SipA\t\r \u00a0is\t\r \u00a0indirect,\t\r \u00a0and\t\r \u00a0the\t\r \u00a0signaling\t\r \u00a0components\t\r \u00a0of\t\r \u00a0this\t\r \u00a0pathway\t\r \u00a0remain\t\r \u00a0largely\t\r \u00a0uncharacterized.\t\r \u00a0While\t\r \u00a0reductionist\t\r \u00a0approaches\t\r \u00a0have\t\r \u00a0been\t\r \u00a0used\t\r \u00a0successfully\t\r \u00a0to\t\r \u00a0identify\t\r \u00a0these\t\r \u00a0signaling\t\r \u00a0events,\t\r \u00a0advances\t\r \u00a0in\t\r \u00a0global\t\r \u00a0proteomics\t\r \u00a0techniques,\t\r \u00a0such\t\r \u00a0as\t\r \u00a0MOC,\t\r \u00a0now\t\r \u00a0make\t\r \u00a0systems-\u00ad\u2010level\t\r \u00a0tracking\t\r \u00a0of\t\r \u00a0protein\t\r \u00a0phosphorylation\t\r \u00a0possible(412,\t\r \u00a0504).\t\r \u00a0These\t\r \u00a0methods\t\r \u00a0in\t\r \u00a0combination\t\r \u00a0with\t\r \u00a0SILAC\t\r \u00a0can\t\r \u00a0resolve\t\r \u00a0protein\t\r \u00a0phosphorylation\t\r \u00a0events\t\r \u00a0in\t\r \u00a0space\t\r \u00a0and\t\r \u00a0time(329).\t\r \u00a0Here\t\r \u00a0we\t\r \u00a0apply\t\r \u00a0quantitative\t\r \u00a0phosphoproteomics\t\r \u00a0to\t\r \u00a0measure\t\r \u00a0more\t\r \u00a0than\t\r \u00a09500\t\r \u00a0phosphorylation\t\r \u00a0events\t\r \u00a0in\t\r \u00a0an\t\r \u00a0epithelial\t\r \u00a0cell\t\r \u00a0line\t\r \u00a0during\t\r \u00a0the\t\r \u00a0initial\t\r \u00a020\t\r \u00a0min\t\r \u00a0following\t\r \u00a0Salmonella\t\r \u00a0infection.\t\r \u00a0Novel\t\r \u00a0host\t\r \u00a0pathways\t\r \u00a0targeted\t\r \u00a0by\t\r \u00a0the\t\r \u00a0bacteria\t\r \u00a0were\t\r \u00a0identified,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0important\t\r \u00a0distinctions\t\r \u00a0between\t\r \u00a0ligand-\u00ad\u2010receptor\t\r \u00a0induced\t\r \u00a0and\t\r \u00a0bacterial\t\r \u00a0induced\t\r \u00a0signaling\t\r \u00a0dynamics.\t\r \u00a0Many\t\r \u00a0of\t\r \u00a0the\t\r \u00a0changes\t\r \u00a0induced\t\r \u00a0by\t\r \u00a0WT\t\r \u00a0S.\t\r \u00a0Typhimurium\t\r \u00a0were\t\r \u00a0functionally\t\r \u00a0validated\t\r \u00a0through\t\r \u00a0the\t\r \u00a0use\t\r \u00a0of\t\r \u00a0a\t\r \u00a0strain\t\r \u00a0mutant\t\r \u00a0for\t\r \u00a0the\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0effector\t\r \u00a0SopB.\t\r \u00a0A\t\r \u00a0novel\t\r \u00a0phosphopeptide\t\r \u00a0in\t\r \u00a0the\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0protein\t\r \u00a0SipB\t\r \u00a0was\t\r \u00a0identified,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0several\t\r \u00a0novel\t\r \u00a0host\t\r \u00a0targets\t\r \u00a0of\t\r \u00a0SopB,\t\r \u00a0reflecting\t\r \u00a0its\t\r \u00a0wide-\u00ad\u2010reaching\t\r \u00a0impact\t\r \u00a0on\t\r \u00a0bacterial-\u00ad\u2010induced\t\r \u00a0signaling.\t\r \u00a0\t\r \u00a0 \t\r \u00a0\t\r \u00a0 110\t\r \u00a0 4.2\t\r \u00a0Experimental\t\r \u00a0procedures\t\r \u00a0 4.2.1\t\r \u00a0Cell\t\r \u00a0culture\t\r \u00a0and\t\r \u00a0Salmonella\t\r \u00a0infection\t\r \u00a0HeLa\t\r \u00a0cells\t\r \u00a0were\t\r \u00a0maintained\t\r \u00a0in\t\r \u00a0DMEM\t\r \u00a0containing\t\r \u00a04500\t\r \u00a0mg\/L\t\r \u00a0glucose\t\r \u00a0and\t\r \u00a04\t\r \u00a0mM\t\r \u00a0L-\u00ad\u2010glutamine\t\r \u00a0(Thermo\t\r \u00a0Fisher\t\r \u00a0Scientific),\t\r \u00a0supplemented\t\r \u00a0with\t\r \u00a010%\t\r \u00a0v\/v\t\r \u00a0qualified\t\r \u00a0FBS\t\r \u00a0(Invitrogen),\t\r \u00a0an\t\r \u00a0additional\t\r \u00a02\t\r \u00a0mM\t\r \u00a0L-\u00ad\u2010glutamine\t\r \u00a0(Thermo\t\r \u00a0Fisher\t\r \u00a0Scientific)\t\r \u00a0and\t\r \u00a00.1\t\r \u00a0U\/L\t\r \u00a0penicillin\t\r \u00a0and\t\r \u00a0streptomycin\t\r \u00a0(Thermo\t\r \u00a0Fisher\t\r \u00a0Scientific).\t\r \u00a0For\t\r \u00a0SILAC\t\r \u00a0labeling,\t\r \u00a0cells\t\r \u00a0were\t\r \u00a0split\t\r \u00a0from\t\r \u00a0normal\t\r \u00a0growth\t\r \u00a0media\t\r \u00a0into\t\r \u00a0arginine\t\r \u00a0and\t\r \u00a0lysine-\u00ad\u2010free\t\r \u00a0DMEM\t\r \u00a0(Caisson\t\r \u00a0Laboratories\t\r \u00a0Inc.)\t\r \u00a0supplemented\t\r \u00a0with\t\r \u00a010%\t\r \u00a0v\/v\t\r \u00a0dialyzed\t\r \u00a0FBS\t\r \u00a0(Invitrogen),\t\r \u00a00.1\t\r \u00a0U\/L\t\r \u00a0penicillin\t\r \u00a0and\t\r \u00a0streptomycin\t\r \u00a0and\t\r \u00a0either\t\r \u00a036.5\t\r \u00a0mg\/mL\t\r \u00a0l-\u00ad\u2010lysine\t\r \u00a0and\t\r \u00a021\t\r \u00a0mg\/mL\t\r \u00a0l-\u00ad\u2010arginine\t\r \u00a0for\t\r \u00a0light\t\r \u00a0labeled\t\r \u00a0cells\t\r \u00a0(Sigma-\u00ad\u2010Aldrich,\t\r \u00a0Oakville,\t\r \u00a0ON),\t\r \u00a037.5\t\r \u00a0mg\/mL\t\r \u00a0D4-\u00ad\u2010lysine\t\r \u00a0and\t\r \u00a021.75\t\r \u00a0mg\/mL\t\r \u00a013C6-\u00ad\u2010arginine\t\r \u00a0for\t\r \u00a0medium\t\r \u00a0labeled\t\r \u00a0cells,\t\r \u00a0and\t\r \u00a038.5\t\r \u00a0mg\/L\t\r \u00a013C615N2-\u00ad\u2010lysine\t\r \u00a0and\t\r \u00a022.25\t\r \u00a0mg\/mL\t\r \u00a013C615N4-\u00ad\u2010arginine\t\r \u00a0for\t\r \u00a0heavy\t\r \u00a0labeled\t\r \u00a0cells\t\r \u00a0(Cambridge\t\r \u00a0Isotope\t\r \u00a0Laboratories,\t\r \u00a0Andover,\t\r \u00a0MA).\t\r \u00a0Cells\t\r \u00a0were\t\r \u00a0then\t\r \u00a0maintained\t\r \u00a0in\t\r \u00a0labeling\t\r \u00a0media\t\r \u00a0for\t\r \u00a0at\t\r \u00a0least\t\r \u00a06\t\r \u00a0cell\t\r \u00a0divisions,\t\r \u00a0as\t\r \u00a0described(330).\t\r \u00a0Eight\t\r \u00a015\t\r \u00a0cm\t\r \u00a0culture\t\r \u00a0dishes\t\r \u00a0of\t\r \u00a0each\t\r \u00a0SILAC\t\r \u00a0label\t\r \u00a0were\t\r \u00a0seeded,\t\r \u00a0each\t\r \u00a0with\t\r \u00a05x106\t\r \u00a0cells,\t\r \u00a0approximately\t\r \u00a040\t\r \u00a0h\t\r \u00a0prior\t\r \u00a0to\t\r \u00a0infection.\t\r \u00a0\t\r \u00a0\t\r \u00a0 Twenty\t\r \u00a0hours\t\r \u00a0prior\t\r \u00a0to\t\r \u00a0infection,\t\r \u00a0SILAC\t\r \u00a0DMEM\t\r \u00a0was\t\r \u00a0removed,\t\r \u00a0cells\t\r \u00a0were\t\r \u00a0washed\t\r \u00a0twice\t\r \u00a0with\t\r \u00a010\t\r \u00a0mL\t\r \u00a0of\t\r \u00a0PBS,\t\r \u00a0and\t\r \u00a016\t\r \u00a0mL\t\r \u00a0serum\t\r \u00a0free\t\r \u00a0and\t\r \u00a0antibiotic\t\r \u00a0free\t\r \u00a0SILAC\t\r \u00a0DMEM\t\r \u00a0were\t\r \u00a0added\t\r \u00a0to\t\r \u00a0each\t\r \u00a0plate.\t\r \u00a0An\t\r \u00a0overnight\t\r \u00a0Salmonella\t\r \u00a0culture\t\r \u00a0(WT\t\r \u00a0S.\t\r \u00a0Typhimurium\t\r \u00a0SL1344\t\r \u00a0or\t\r \u00a0S.\t\r \u00a0Typhimurium\t\r \u00a0SL1344\t\r \u00a0\u0394sopB(461))\t\r \u00a0was\t\r \u00a0subcultured\t\r \u00a0(1:33)\t\r \u00a0in\t\r \u00a0LB\t\r \u00a0broth\t\r \u00a0for\t\r \u00a03\t\r \u00a0h.\t\r \u00a0The\t\r \u00a0bacterial\t\r \u00a0inoculum\t\r \u00a0was\t\r \u00a0prepared\t\r \u00a0by\t\r \u00a0pelleting\t\r \u00a0bacteria\t\r \u00a0at\t\r \u00a010,000\t\r \u00a0rcf\t\r \u00a0for\t\r \u00a02\t\r \u00a0min\t\r \u00a0at\t\r \u00a0RT.\t\r \u00a0Bacterial\t\r \u00a0pellets\t\r \u00a0were\t\r \u00a0re-\u00ad\u2010suspended\t\r \u00a0in\t\r \u00a0serum-\u00ad\u2010free,\t\r \u00a0antibiotic-\u00ad\u2010free\t\r \u00a0DMEM\t\r \u00a0and\t\r \u00a0HeLa\t\r \u00a0cells\t\r \u00a0were\t\r \u00a0infected\t\r \u00a0at\t\r \u00a0a\t\r \u00a0multiplicity\t\r \u00a0of\t\r \u00a0infection\t\r \u00a0 \t\r \u00a0\t\r \u00a0 111\t\r \u00a0 (MOI)\t\r \u00a0of\t\r \u00a0200\t\r \u00a0and\t\r \u00a0incubated\t\r \u00a0at\t\r \u00a037\u00b0C\t\r \u00a0and\t\r \u00a05%\t\r \u00a0CO2\t\r \u00a0(light\t\r \u00a0labeled\t\r \u00a0cells\t\r \u00a0were\t\r \u00a0mock\t\r \u00a0infected\t\r \u00a0in\t\r \u00a0media\t\r \u00a0only).\t\r \u00a0At\t\r \u00a0the\t\r \u00a0desired\t\r \u00a0infection\t\r \u00a0times,\t\r \u00a0cells\t\r \u00a0were\t\r \u00a0removed\t\r \u00a0from\t\r \u00a0the\t\r \u00a0incubator\t\r \u00a0and\t\r \u00a0immediately\t\r \u00a0placed\t\r \u00a0on\t\r \u00a0ice.\t\r \u00a0Media\t\r \u00a0was\t\r \u00a0removed\t\r \u00a0and\t\r \u00a0plates\t\r \u00a0were\t\r \u00a0washed\t\r \u00a0three\t\r \u00a0times\t\r \u00a0with\t\r \u00a010\t\r \u00a0mL\t\r \u00a0cold\t\r \u00a0PBS\t\r \u00a0and\t\r \u00a0harvested\t\r \u00a0with\t\r \u00a0a\t\r \u00a0scraper.\t\r \u00a0Cells\t\r \u00a0carrying\t\r \u00a0the\t\r \u00a0same\t\r \u00a0SILAC\t\r \u00a0label\t\r \u00a0were\t\r \u00a0pooled,\t\r \u00a0pelleted\t\r \u00a0for\t\r \u00a04\t\r \u00a0min\t\r \u00a0at\t\r \u00a0600\t\r \u00a0rcf\t\r \u00a0at\t\r \u00a04\u00b0C\t\r \u00a0and\t\r \u00a0re-\u00ad\u2010suspended\t\r \u00a0in\t\r \u00a03.2\t\r \u00a0mL\t\r \u00a0cold\t\r \u00a0lysis\t\r \u00a0buffer\t\r \u00a0(1\t\r \u00a0mM\t\r \u00a0Na3VO4,\t\r \u00a010\t\r \u00a0mM\t\r \u00a0NaF,\t\r \u00a05\t\r \u00a0mM\t\r \u00a0Na4P2O7,\t\r \u00a020\t\r \u00a0mM\t\r \u00a0\u03b2-\u00ad\u2010glycerophosphate,\t\r \u00a00.5\t\r \u00a0mM\t\r \u00a0pervanadate,\t\r \u00a0100\t\r \u00a0\u00b5M\t\r \u00a0deltamethrin,\t\r \u00a0100\t\r \u00a0\u00b5M\t\r \u00a0calyculin\t\r \u00a0A,\t\r \u00a0and\t\r \u00a0Complete\t\r \u00a0protease\t\r \u00a0inhibitor\t\r \u00a0cocktail\t\r \u00a0(Roche\t\r \u00a0Diagnostics)\t\r \u00a0in\t\r \u00a0PBS).\t\r \u00a0Cells\t\r \u00a0were\t\r \u00a0lysed\t\r \u00a0on\t\r \u00a0ice\t\r \u00a0by\t\r \u00a0five\t\r \u00a0or\t\r \u00a0six\t\r \u00a0passages\t\r \u00a0through\t\r \u00a0a\t\r \u00a022\t\r \u00a0G\t\r \u00a0needle,\t\r \u00a0and\t\r \u00a0nuclei\t\r \u00a0were\t\r \u00a0pelleted\t\r \u00a0by\t\r \u00a0centrifugation\t\r \u00a0for\t\r \u00a04\t\r \u00a0min\t\r \u00a0at\t\r \u00a0600\t\r \u00a0rcf\t\r \u00a0at\t\r \u00a04\u00b0C.\t\r \u00a0The\t\r \u00a0supernatant\t\r \u00a0was\t\r \u00a0retained\t\r \u00a0and\t\r \u00a0the\t\r \u00a0pellet\t\r \u00a0(nuclear\t\r \u00a0fraction)\t\r \u00a0was\t\r \u00a0re-\u00ad\u2010suspended\t\r \u00a0in\t\r \u00a01.6\t\r \u00a0mL\t\r \u00a0Digestion\t\r \u00a0Buffer\t\r \u00a0(1%\t\r \u00a0w\/v\t\r \u00a0sodium\t\r \u00a0deoxycholate\/50\t\r \u00a0mM\t\r \u00a0NH4HCO3)\t\r \u00a0and\t\r \u00a0immediately\t\r \u00a0heated\t\r \u00a0at\t\r \u00a099\u00b0C\t\r \u00a0for\t\r \u00a05\t\r \u00a0min.\t\r \u00a0Membranes\t\r \u00a0were\t\r \u00a0pelleted\t\r \u00a0from\t\r \u00a0the\t\r \u00a0supernatant\t\r \u00a0for\t\r \u00a030\t\r \u00a0min\t\r \u00a0at\t\r \u00a016,000\t\r \u00a0rcf\t\r \u00a0at\t\r \u00a04\u00b0C\t\r \u00a0and\t\r \u00a0the\t\r \u00a0supernatant\t\r \u00a0was\t\r \u00a0retained.\t\r \u00a0The\t\r \u00a0pellet\t\r \u00a0and\t\r \u00a0supernatant\t\r \u00a0from\t\r \u00a0this\t\r \u00a0step\t\r \u00a0were\t\r \u00a0mixed\t\r \u00a0with\t\r \u00a0800\t\r \u00a0\u00b5L\t\r \u00a0and\t\r \u00a01.6\t\r \u00a0mL\t\r \u00a0of\t\r \u00a0Digestion\t\r \u00a0Buffer\t\r \u00a0respectively\t\r \u00a0and\t\r \u00a0both\t\r \u00a0were\t\r \u00a0heated\t\r \u00a0at\t\r \u00a099\u00b0C\t\r \u00a0for\t\r \u00a05\t\r \u00a0min.\t\r \u00a0Samples\t\r \u00a0were\t\r \u00a0removed\t\r \u00a0and\t\r \u00a0cooled\t\r \u00a0to\t\r \u00a0RT,\t\r \u00a0MgCl2\t\r \u00a0was\t\r \u00a0added\t\r \u00a0to\t\r \u00a0a\t\r \u00a0final\t\r \u00a0concentration\t\r \u00a0of\t\r \u00a01.5\t\r \u00a0mM,\t\r \u00a0and\t\r \u00a02.5x10-\u00ad\u20103\t\r \u00a0U\/\u00b5L\t\r \u00a0benzonase\t\r \u00a0(Novagen)\t\r \u00a0were\t\r \u00a0added\t\r \u00a0to\t\r \u00a0cleave\t\r \u00a0DNA\t\r \u00a0and\t\r \u00a0decrease\t\r \u00a0viscosity.\t\r \u00a0Protein\t\r \u00a0concentrations\t\r \u00a0were\t\r \u00a0determined\t\r \u00a0for\t\r \u00a0each\t\r \u00a0sample\t\r \u00a0using\t\r \u00a0a\t\r \u00a0standard\t\r \u00a0Bradford\t\r \u00a0Assay\t\r \u00a0(Pierce).\t\r \u00a0Eight\t\r \u00a0milligrams\t\r \u00a0of\t\r \u00a0protein\t\r \u00a0from\t\r \u00a0each\t\r \u00a0nuclear\t\r \u00a0fraction\t\r \u00a0were\t\r \u00a0combined,\t\r \u00a08\t\r \u00a0mg\t\r \u00a0from\t\r \u00a0each\t\r \u00a0cytosolic\t\r \u00a0fraction\t\r \u00a0were\t\r \u00a0combined,\t\r \u00a0and\t\r \u00a02\t\r \u00a0mg\t\r \u00a0from\t\r \u00a0each\t\r \u00a0membrane\t\r \u00a0fractions\t\r \u00a0were\t\r \u00a0combined.\t\r \u00a0\t\r \u00a0\t\r \u00a0 \t\r \u00a0\t\r \u00a0 112\t\r \u00a0 4.2.2\t\r \u00a0Sample\t\r \u00a0generation\t\r \u00a0for\t\r \u00a0phosphoproteomics\t\r \u00a0Samples\t\r \u00a0were\t\r \u00a0subjected\t\r \u00a0to\t\r \u00a0tryptic\t\r \u00a0digest,\t\r \u00a0desalting,\t\r \u00a0in-\u00ad\u2010solution\t\r \u00a0IEF,\t\r \u00a0and\t\r \u00a0phosphopeptide\t\r \u00a0enrichment\t\r \u00a0conditions\t\r \u00a0exactly\t\r \u00a0as\t\r \u00a0previously\t\r \u00a0described\t\r \u00a0(Chapter\t\r \u00a03).\t\r \u00a0\t\r \u00a0\t\r \u00a0 4.2.3\t\r \u00a0Mass\t\r \u00a0spectrometry\t\r \u00a0For\t\r \u00a0LC-\u00ad\u2010MSn,\t\r \u00a0samples\t\r \u00a0were\t\r \u00a0analyzed\t\r \u00a0on\t\r \u00a0a\t\r \u00a0LTQ-\u00ad\u2010OrbitrapXL\t\r \u00a0(Thermo\t\r \u00a0Fisher\t\r \u00a0Scientific)\t\r \u00a0coupled\t\r \u00a0on-\u00ad\u2010line\t\r \u00a0to\t\r \u00a0an\t\r \u00a0Agilent\t\r \u00a01100\t\r \u00a0Series\t\r \u00a0nanoflow\t\r \u00a0HPLC\t\r \u00a0instrument\t\r \u00a0using\t\r \u00a0a\t\r \u00a0nanospray\t\r \u00a0ionization\t\r \u00a0source\t\r \u00a0(Proxeon\t\r \u00a0Biosystems)\t\r \u00a0as\t\r \u00a0previously\t\r \u00a0described\t\r \u00a0(Chapter\t\r \u00a02).\t\r \u00a0\t\r \u00a0 4.2.4\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0data\t\r \u00a0analysis\t\r \u00a0and\t\r \u00a0clustering\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0data\t\r \u00a0were\t\r \u00a0analyzed\t\r \u00a0using\t\r \u00a0MaxQuant\t\r \u00a0software\t\r \u00a0(v1.0.1.13)\t\r \u00a0as\t\r \u00a0previously\t\r \u00a0described,\t\r \u00a0except\t\r \u00a0that\t\r \u00a0only\t\r \u00a0N-\u00ad\u2010terminal\t\r \u00a0acetylation\t\r \u00a0and\t\r \u00a0phosphorylation\t\r \u00a0of\t\r \u00a0serine,\t\r \u00a0threonine,\t\r \u00a0and\t\r \u00a0tyrosine\t\r \u00a0residues\t\r \u00a0were\t\r \u00a0selected\t\r \u00a0as\t\r \u00a0variable\t\r \u00a0modifications(334).\t\r \u00a0All\t\r \u00a0peptide\t\r \u00a0and\t\r \u00a0protein\t\r \u00a0data\t\r \u00a0from\t\r \u00a0this\t\r \u00a0study\t\r \u00a0are\t\r \u00a0publicly\t\r \u00a0available\t\r \u00a0at\t\r \u00a0www.phosphonet.ca.\t\r \u00a0FCM\t\r \u00a0clustering\t\r \u00a0was\t\r \u00a0done\t\r \u00a0using\t\r \u00a0the\t\r \u00a0MFuzz\t\r \u00a0toolbox\t\r \u00a0with\t\r \u00a0c\t\r \u00a0(number\t\r \u00a0of\t\r \u00a0clusters)\t\r \u00a0and\t\r \u00a0m\t\r \u00a0(fuzzification\t\r \u00a0parameter)\t\r \u00a0values\t\r \u00a0of\t\r \u00a06\t\r \u00a0and\t\r \u00a02(505).\t\r \u00a0Clustering\t\r \u00a0for\t\r \u00a0GO\t\r \u00a0analysis\t\r \u00a0was\t\r \u00a0done\t\r \u00a0using\t\r \u00a0the\t\r \u00a0DAVID\t\r \u00a0Functional\t\r \u00a0Annotation\t\r \u00a0Tool(506,\t\r \u00a0507).\t\r \u00a0Common\t\r \u00a0motifs\t\r \u00a0were\t\r \u00a0searched\t\r \u00a0using\t\r \u00a0Motif-\u00ad\u2010X,\t\r \u00a0and\t\r \u00a0default\t\r \u00a0settings\t\r \u00a0were\t\r \u00a0used\t\r \u00a0except\t\r \u00a0that\t\r \u00a0MS\/MS\t\r \u00a0was\t\r \u00a0selected\t\r \u00a0as\t\r \u00a0a\t\r \u00a0foreground\t\r \u00a0format\t\r \u00a0and\t\r \u00a0phosphorylated\t\r \u00a0serine\t\r \u00a0was\t\r \u00a0selected\t\r \u00a0as\t\r \u00a0the\t\r \u00a0central\t\r \u00a0character(508).\t\r \u00a0Predictions\t\r \u00a0of\t\r \u00a0up-\u00ad\u2010stream\t\r \u00a0kinases\t\r \u00a0were\t\r \u00a0performed\t\r \u00a0using\t\r \u00a0data\t\r \u00a0in\t\r \u00a0PhosphoNET,\t\r \u00a0and\t\r \u00a0the\t\r \u00a0evolutionary\t\r \u00a0 \t\r \u00a0\t\r \u00a0 113\t\r \u00a0 conservation\t\r \u00a0and\t\r \u00a0phosphorylating\t\r \u00a0kinase\t\r \u00a0data\t\r \u00a0are\t\r \u00a0also\t\r \u00a0publicly\t\r \u00a0available\t\r \u00a0in\t\r \u00a0PhosphoNET.\t\r \u00a0\t\r \u00a0 4.2.5\t\r \u00a0High-\u00ad\u2010throughput\t\r \u00a0western\t\r \u00a0blotting\t\r \u00a0Custom\t\r \u00a0Kinetworks\u2122\t\r \u00a0KCPS\t\r \u00a0multi-\u00ad\u2010immunoblotting\t\r \u00a0analyses\t\r \u00a0were\t\r \u00a0performed\t\r \u00a0as\t\r \u00a0described\t\r \u00a0previously\t\r \u00a0(www.kinexus.ca)(509)\t\r \u00a0using\t\r \u00a0300\t\r \u00a0\u00b5g\t\r \u00a0of\t\r \u00a0detergent-\u00ad\u2010solubilized\t\r \u00a0HeLa\t\r \u00a0lysates\t\r \u00a0prepared\t\r \u00a0as\t\r \u00a0above.\t\r \u00a0The\t\r \u00a0Kinetworks\u2122\t\r \u00a0analysis\t\r \u00a0involves\t\r \u00a0resolution\t\r \u00a0of\t\r \u00a0proteins\t\r \u00a0in\t\r \u00a0a\t\r \u00a0single\t\r \u00a0lysate\t\r \u00a0sample\t\r \u00a0by\t\r \u00a0SDS-\u00ad\u2010PAGE\t\r \u00a0and\t\r \u00a0subsequent\t\r \u00a0immunoblotting\t\r \u00a0overnight\t\r \u00a0at\t\r \u00a04\u00b0C\t\r \u00a0with\t\r \u00a01\t\r \u00a0to\t\r \u00a03\t\r \u00a0primary\t\r \u00a0phosphosite-\u00ad\u2010specific\t\r \u00a0antibodies\t\r \u00a0per\t\r \u00a0channel\t\r \u00a0in\t\r \u00a0a\t\r \u00a020-\u00ad\u2010lane\t\r \u00a0Immunetics\t\r \u00a0multiblotter.\t\r \u00a0Phosphosite\t\r \u00a0antibodies\t\r \u00a0were\t\r \u00a0typically\t\r \u00a0sourced\t\r \u00a0from\t\r \u00a0Invitrogen\t\r \u00a0(Carlsbad,\t\r \u00a0CA),\t\r \u00a0Cell\t\r \u00a0Signaling\t\r \u00a0Technologies\t\r \u00a0(Beverly,\t\r \u00a0MA)\t\r \u00a0and\t\r \u00a0Millipore\t\r \u00a0(Temecula,\t\r \u00a0CA).\t\r \u00a0The\t\r \u00a0antibody\t\r \u00a0mixtures\t\r \u00a0were\t\r \u00a0carefully\t\r \u00a0selected\t\r \u00a0to\t\r \u00a0avoid\t\r \u00a0overlapping\t\r \u00a0cross-\u00ad\u2010reactivity\t\r \u00a0with\t\r \u00a0target\t\r \u00a0proteins.\t\r \u00a0The\t\r \u00a0membranes\t\r \u00a0were\t\r \u00a0later\t\r \u00a0rinsed\t\r \u00a0with\t\r \u00a0TBST\t\r \u00a0buffer\t\r \u00a0(50\t\r \u00a0mM\t\r \u00a0Tris\t\r \u00a0base,\t\r \u00a0150\t\r \u00a0mM\t\r \u00a0NaCl,\t\r \u00a00.5%\t\r \u00a0Triton\t\r \u00a0X-\u00ad\u2010100\t\r \u00a0(v\/v),\t\r \u00a0pH\t\r \u00a07.4)\t\r \u00a0and\t\r \u00a0then\t\r \u00a0incubated\t\r \u00a0with\t\r \u00a0the\t\r \u00a0relevant\t\r \u00a0horseradish\t\r \u00a0peroxidase\t\r \u00a0conjugated\t\r \u00a0secondary\t\r \u00a0antibodies\t\r \u00a0for\t\r \u00a045\t\r \u00a0min\t\r \u00a0at\t\r \u00a0RT.\t\r \u00a0The\t\r \u00a0immunoblots\t\r \u00a0were\t\r \u00a0developed\t\r \u00a0with\t\r \u00a0enhanced\t\r \u00a0chemiluminescence\t\r \u00a0(ECL)\t\r \u00a0Plus\t\r \u00a0reagent\t\r \u00a0(Amersham,\t\r \u00a0Arlington\t\r \u00a0Heights,\t\r \u00a0IL),\t\r \u00a0and\t\r \u00a0signals\t\r \u00a0were\t\r \u00a0captured\t\r \u00a0by\t\r \u00a0a\t\r \u00a0Fluor-\u00ad\u2010S\t\r \u00a0MultiImager\t\r \u00a0and\t\r \u00a0quantified\t\r \u00a0using\t\r \u00a0Quantity\t\r \u00a0One\t\r \u00a0software\t\r \u00a0(Bio-\u00ad\u2010Rad,\t\r \u00a0Hercules,\t\r \u00a0CA).\t\r \u00a0Background\t\r \u00a0was\t\r \u00a0less\t\r \u00a0than\t\r \u00a0100\t\r \u00a0counts\t\r \u00a0per\t\r \u00a0minute\t\r \u00a0for\t\r \u00a0these\t\r \u00a0analyses.\t\r \u00a0\t\r \u00a0 \t\r \u00a0\t\r \u00a0 114\t\r \u00a0 4.3\t\r \u00a0Results\t\r \u00a0 4.3.1\t\r \u00a0Quantitative\t\r \u00a0phosphoproteomic\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0signaling\t\r \u00a0during\t\r \u00a0 Salmonella\t\r \u00a0invasion\t\r \u00a0For\t\r \u00a0phosphoproteomic\t\r \u00a0analysis,\t\r \u00a0three\t\r \u00a0HeLa\t\r \u00a0cell\t\r \u00a0populations\t\r \u00a0were\t\r \u00a0labeled\t\r \u00a0with\t\r \u00a0light,\t\r \u00a0medium,\t\r \u00a0and\t\r \u00a0heavy\t\r \u00a0isotopologs\t\r \u00a0of\t\r \u00a0lysine\t\r \u00a0and\t\r \u00a0arginine\t\r \u00a0for\t\r \u00a0SILAC\t\r \u00a0quantitation\t\r \u00a0(Figure\t\r \u00a04.1).\t\r \u00a0Light\t\r \u00a0labeled\t\r \u00a0populations\t\r \u00a0were\t\r \u00a0mock\t\r \u00a0infected,\t\r \u00a0while\t\r \u00a0medium\t\r \u00a0and\t\r \u00a0heavy\t\r \u00a0labeled\t\r \u00a0populations\t\r \u00a0were\t\r \u00a0infected\t\r \u00a0with\t\r \u00a0WT\t\r \u00a0or\t\r \u00a0\u0394sopB\t\r \u00a0strains\t\r \u00a0for\t\r \u00a0the\t\r \u00a0various\t\r \u00a0times\t\r \u00a0indicated.\t\r \u00a0Cells\t\r \u00a0were\t\r \u00a0lysed,\t\r \u00a0separated\t\r \u00a0into\t\r \u00a0nuclear,\t\r \u00a0membrane\t\r \u00a0and\t\r \u00a0cytosolic\t\r \u00a0fractions,\t\r \u00a0and\t\r \u00a0solubilized\t\r \u00a0in\t\r \u00a0detergent.\t\r \u00a0Equal\t\r \u00a0masses\t\r \u00a0of\t\r \u00a0protein\t\r \u00a0from\t\r \u00a0each\t\r \u00a0SILAC\t\r \u00a0label\/fraction\t\r \u00a0were\t\r \u00a0combined,\t\r \u00a0digested\t\r \u00a0with\t\r \u00a0trypsin,\t\r \u00a0fractionated\t\r \u00a0by\t\r \u00a0in-\u00ad\u2010solution\t\r \u00a0IEF,\t\r \u00a0and\t\r \u00a0phosphopeptides\t\r \u00a0were\t\r \u00a0enriched\t\r \u00a0by\t\r \u00a0MOC(504).\t\r \u00a0Biological\t\r \u00a0replicates\t\r \u00a0of\t\r \u00a0each\t\r \u00a0experiment\t\r \u00a0each\t\r \u00a0yielded\t\r \u00a030\t\r \u00a0samples\t\r \u00a0for\t\r \u00a0LC-\u00ad\u2010MSn.\t\r \u00a0\t\r \u00a0 Altogether,\t\r \u00a01973\t\r \u00a0phosphorylated\t\r \u00a0proteins\t\r \u00a0were\t\r \u00a0identified\t\r \u00a0with\t\r \u00a0a\t\r \u00a0false\t\r \u00a0discovery\t\r \u00a0rate\t\r \u00a0of\t\r \u00a01%,\t\r \u00a0corresponding\t\r \u00a0to\t\r \u00a06893\t\r \u00a0non-\u00ad\u2010redundant\t\r \u00a0phosphopeptides\t\r \u00a0and\t\r \u00a09508\t\r \u00a0non-\u00ad\u2010redundant\t\r \u00a0phosphorylation\t\r \u00a0sites\t\r \u00a0(www.phosphonet.ca).\t\r \u00a0Significantly,\t\r \u00a0the\t\r \u00a0dynamic\t\r \u00a0profiles\t\r \u00a0measured\t\r \u00a0appeared\t\r \u00a0to\t\r \u00a0be\t\r \u00a0exceptionally\t\r \u00a0accurate,\t\r \u00a0as\t\r \u00a0the\t\r \u00a0average\t\r \u00a0coefficient\t\r \u00a0of\t\r \u00a0variation\t\r \u00a0(!CV)\t\r \u00a0across\t\r \u00a0biological\t\r \u00a0replicates\t\r \u00a0was\t\r \u00a0<15%.\t\r \u00a0More\t\r \u00a0than\t\r \u00a085%\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0were\t\r \u00a0quantified\t\r \u00a0in\t\r \u00a0at\t\r \u00a0least\t\r \u00a0one\t\r \u00a0experiment,\t\r \u00a0and\t\r \u00a061,\t\r \u00a043\t\r \u00a0and\t\r \u00a049%\t\r \u00a0of\t\r \u00a0these\t\r \u00a0were\t\r \u00a0identified\t\r \u00a0in\t\r \u00a0the\t\r \u00a0nuclear,\t\r \u00a0membrane\t\r \u00a0and\t\r \u00a0cytosolic\t\r \u00a0fractions\t\r \u00a0respectively.\t\r \u00a0The\t\r \u00a0distribution\t\r \u00a0of\t\r \u00a0events\t\r \u00a0between\t\r \u00a0pS,\t\r \u00a0pT\t\r \u00a0and\t\r \u00a0pY\t\r \u00a0was\t\r \u00a082:15:3,\t\r \u00a0similar\t\r \u00a0to\t\r \u00a0a\t\r \u00a0previous\t\r \u00a0report(297)\t\r \u00a0and\t\r \u00a0highly\t\r \u00a0correlated\t\r \u00a0with\t\r \u00a0the\t\r \u00a080:16:4\t\r \u00a0ratio\t\r \u00a0present\t\r \u00a0in\t\r \u00a0PhosphoNET\t\r \u00a0(www.phosphonet.ca).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 115\t\r \u00a0 Figure\t\r \u00a04.1\t\r \u00a0Flow\t\r \u00a0diagram\t\r \u00a0outlining\t\r \u00a0phosphoproteomics\t\r \u00a0method\t\r \u00a0for\t\r \u00a0Salmonella\t\r \u00a0infection\t\r \u00a0 \t\r \u00a0Pools\t\r \u00a0of\t\r \u00a0triple-\u00ad\u2010SILAC\t\r \u00a0labeled\t\r \u00a0HeLa\t\r \u00a0cells\t\r \u00a0were\t\r \u00a0either\t\r \u00a0mock\t\r \u00a0infected\t\r \u00a0(N-\u00ad\u2010Lys,\t\r \u00a0N-\u00ad\u2010Arg),\t\r \u00a0or\t\r \u00a0infected\t\r \u00a0with\t\r \u00a0WT\t\r \u00a0S.\t\r \u00a0Typhimurium\t\r \u00a0for\t\r \u00a02\t\r \u00a0(4-\u00ad\u2010Lys,\t\r \u00a06-\u00ad\u2010Arg)\t\r \u00a0and\t\r \u00a020\t\r \u00a0(8-\u00ad\u2010Lys,\t\r \u00a010-\u00ad\u2010Arg)\t\r \u00a0min\t\r \u00a0(Experiment\t\r \u00a01),\t\r \u00a0or\t\r \u00a05\t\r \u00a0(4-\u00ad\u2010Lys,\t\r \u00a06-\u00ad\u2010Arg)\t\r \u00a0and\t\r \u00a010\t\r \u00a0(8-\u00ad\u2010Lys,\t\r \u00a010-\u00ad\u2010Arg)\t\r \u00a0min\t\r \u00a0(Experiment\t\r \u00a02).\t\r \u00a0For\t\r \u00a0Experiment\t\r \u00a03,\t\r \u00a0cells\t\r \u00a0were\t\r \u00a0infected\t\r \u00a0with\t\r \u00a0\u0394sopB\t\r \u00a0S.\t\r \u00a0Typhimurium\t\r \u00a0for\t\r \u00a05\t\r \u00a0(4-\u00ad\u2010Lys,\t\r \u00a06-\u00ad\u2010Arg)\t\r \u00a0and\t\r \u00a020\t\r \u00a0(8-\u00ad\u2010Lys,\t\r \u00a010-\u00ad\u2010Arg)\t\r \u00a0min.\t\r \u00a0All\t\r \u00a0cells\t\r \u00a0were\t\r \u00a0mechanically\t\r \u00a0lysed\t\r \u00a0and\t\r \u00a0separated\t\r \u00a0by\t\r \u00a0centrifugation\t\r \u00a0into\t\r \u00a0nuclear,\t\r \u00a0membrane\t\r \u00a0and\t\r \u00a0cytosolic\t\r \u00a0fractions.\t\r \u00a0Fractions\t\r \u00a0were\t\r \u00a0solubilized\t\r \u00a0in\t\r \u00a0detergent,\t\r \u00a0and\t\r \u00a0equal\t\r \u00a0protein\t\r \u00a0amounts\t\r \u00a0were\t\r \u00a0combined\t\r \u00a0for\t\r \u00a0each\t\r \u00a0SILAC\t\r \u00a0label.\t\r \u00a0Tryptic\t\r \u00a0peptides\t\r \u00a0were\t\r \u00a0fractionated\t\r \u00a0by\t\r \u00a0in-\u00ad\u2010solution\t\r \u00a0IEF\t\r \u00a0and\t\r \u00a0phosphopeptides\t\r \u00a0were\t\r \u00a0enriched\t\r \u00a0using\t\r \u00a0MOC.\t\r \u00a0For\t\r \u00a0each\t\r \u00a0of\t\r \u00a0Experiments\t\r \u00a01,\t\r \u00a02,\t\r \u00a0and\t\r \u00a03\t\r \u00a0the\t\r \u00a0resulting\t\r \u00a030\t\r \u00a0fractions\t\r \u00a0were\t\r \u00a0analyzed\t\r \u00a0by\t\r \u00a0LC-\u00ad\u2010MSn.\t\r \u00a0\t\r \u00a0 At\t\r \u00a0least\t\r \u00a024%\t\r \u00a0of\t\r \u00a0detected\t\r \u00a0phosphopeptides\t\r \u00a0were\t\r \u00a0differentially\t\r \u00a0regulated\t\r \u00a0(P<.01)\t\r \u00a0during\t\r \u00a0infection.\t\r \u00a0Fuzzy\t\r \u00a0c-\u00ad\u2010means\t\r \u00a0clustering\t\r \u00a0of\t\r \u00a0the\t\r \u00a0regulated\t\r \u00a0profiles\t\r \u00a0 \t\r \u00a0\t\r \u00a0 116\t\r \u00a0 generated\t\r \u00a0four\t\r \u00a0clusters\t\r \u00a0containing\t\r \u00a0up-\u00ad\u2010regulated\t\r \u00a0phosphopeptides\t\r \u00a0and\t\r \u00a0two\t\r \u00a0containing\t\r \u00a0down-\u00ad\u2010regulated\t\r \u00a0phosphopeptides\t\r \u00a0(Figure\t\r \u00a04.2A).\t\r \u00a0The\t\r \u00a0clusters\t\r \u00a0of\t\r \u00a0up-\u00ad\u2010regulated\t\r \u00a0phosphopeptides\t\r \u00a0are\t\r \u00a0dramatically\t\r \u00a0different,\t\r \u00a0peaking\t\r \u00a0or\t\r \u00a0beginning\t\r \u00a0to\t\r \u00a0increase\t\r \u00a0in\t\r \u00a0abundance\t\r \u00a0at\t\r \u00a0early\t\r \u00a0or\t\r \u00a0late\t\r \u00a0time\t\r \u00a0points.\t\r \u00a0Similarly,\t\r \u00a0the\t\r \u00a0clusters\t\r \u00a0containing\t\r \u00a0down-\u00ad\u2010regulated\t\r \u00a0phosphopeptides\t\r \u00a0begin\t\r \u00a0to\t\r \u00a0decrease\t\r \u00a0either\t\r \u00a0early\t\r \u00a0or\t\r \u00a0quite\t\r \u00a0late,\t\r \u00a0indicating\t\r \u00a0stark\t\r \u00a0differences\t\r \u00a0in\t\r \u00a0phosphopeptide\t\r \u00a0profiles\t\r \u00a0across\t\r \u00a0the\t\r \u00a0dataset.\t\r \u00a0There\t\r \u00a0were\t\r \u00a0striking\t\r \u00a0differences\t\r \u00a0among\t\r \u00a0the\t\r \u00a0Gene\t\r \u00a0Ontology\t\r \u00a0(GO)\t\r \u00a0terms\t\r \u00a0enriched\t\r \u00a0in\t\r \u00a0the\t\r \u00a0clustered\t\r \u00a0up-\u00ad\u2010\t\r \u00a0and\t\r \u00a0down-\u00ad\u2010regulated\t\r \u00a0phosphoproteins:\t\r \u00a0terms\t\r \u00a0for\t\r \u00a0regulation\t\r \u00a0of\t\r \u00a0apoptosis,\t\r \u00a0transmembrane\t\r \u00a0transport,\t\r \u00a0nuclear\t\r \u00a0organization\t\r \u00a0and\t\r \u00a0cell\t\r \u00a0proliferation\t\r \u00a0were\t\r \u00a0enriched\t\r \u00a0among\t\r \u00a0proteins\t\r \u00a0with\t\r \u00a0up-\u00ad\u2010regulated\t\r \u00a0phosphorylation\t\r \u00a0sites,\t\r \u00a0while\t\r \u00a0terms\t\r \u00a0for\t\r \u00a0cytoskeleton\t\r \u00a0organization,\t\r \u00a0protein\t\r \u00a0complex\t\r \u00a0assembly,\t\r \u00a0and\t\r \u00a0cell\t\r \u00a0polarity\t\r \u00a0were\t\r \u00a0enriched\t\r \u00a0among\t\r \u00a0proteins\t\r \u00a0with\t\r \u00a0down-\u00ad\u2010regulated\t\r \u00a0phosphorylation\t\r \u00a0sites\t\r \u00a0(Figure\t\r \u00a04.2B).\t\r \u00a0 Overall\t\r \u00a0trends\t\r \u00a0within\t\r \u00a0the\t\r \u00a0nuclear,\t\r \u00a0membrane,\t\r \u00a0and\t\r \u00a0cytosolic\t\r \u00a0fractions\t\r \u00a0were\t\r \u00a0also\t\r \u00a0considered.\t\r \u00a0While\t\r \u00a0the\t\r \u00a0percentage\t\r \u00a0of\t\r \u00a0regulated\t\r \u00a0phosphopeptides\t\r \u00a0was\t\r \u00a0similar\t\r \u00a0(22%,\t\r \u00a018%,\t\r \u00a020%)\t\r \u00a0across\t\r \u00a0each\t\r \u00a0fraction,\t\r \u00a0down-\u00ad\u2010regulated\t\r \u00a0phosphopeptides\t\r \u00a0were\t\r \u00a0over-\u00ad\u2010represented\t\r \u00a0in\t\r \u00a0the\t\r \u00a0membrane\t\r \u00a0and\t\r \u00a0nuclear\t\r \u00a0fractions\t\r \u00a0(Figure\t\r \u00a04.2C).\t\r \u00a0Interestingly,\t\r \u00a0while\t\r \u00a0quicker\t\r \u00a0dynamics\t\r \u00a0might\t\r \u00a0be\t\r \u00a0expected\t\r \u00a0in\t\r \u00a0the\t\r \u00a0membrane\t\r \u00a0and\t\r \u00a0cytosolic\t\r \u00a0fractions\t\r \u00a0(due\t\r \u00a0to\t\r \u00a0cascades\t\r \u00a0initiating\t\r \u00a0at\t\r \u00a0the\t\r \u00a0cell\t\r \u00a0periphery\t\r \u00a0and\t\r \u00a0sites\t\r \u00a0of\t\r \u00a0invasion),\t\r \u00a0overall\t\r \u00a0rates\t\r \u00a0are\t\r \u00a0similar\t\r \u00a0across\t\r \u00a0each\t\r \u00a0fraction.\t\r \u00a0This\t\r \u00a0may\t\r \u00a0be\t\r \u00a0due\t\r \u00a0to\t\r \u00a0the\t\r \u00a0fact\t\r \u00a0that\t\r \u00a0the\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0pool\t\r \u00a0of\t\r \u00a0effectors\t\r \u00a0are\t\r \u00a0injected\t\r \u00a0into\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0very\t\r \u00a0quickly,\t\r \u00a0and\t\r \u00a0several\t\r \u00a0have\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0directly\t\r \u00a0target\t\r \u00a0host\t\r \u00a0proteins\t\r \u00a0in\t\r \u00a0the\t\r \u00a0cytosol\t\r \u00a0and\t\r \u00a0nucleus(143,\t\r \u00a0183,\t\r \u00a0510).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 117\t\r \u00a0 Figure\t\r \u00a04.2\t\r \u00a0Global\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0phosphoproteomics\t\r \u00a0data\t\r \u00a0 \t\r \u00a0 \t\r \u00a0\t\r \u00a0 118\t\r \u00a0 A)\t\r \u00a0Clustering\t\r \u00a0of\t\r \u00a0all\t\r \u00a0dynamic\t\r \u00a0phosphopeptide\t\r \u00a0profiles\t\r \u00a0(P<.01)\t\r \u00a0by\t\r \u00a0FCM\t\r \u00a0clustering.\t\r \u00a0Each\t\r \u00a0profile\t\r \u00a0is\t\r \u00a0color-\u00ad\u2010coded\t\r \u00a0according\t\r \u00a0to\t\r \u00a0membership\t\r \u00a0values\t\r \u00a0shown\t\r \u00a0on\t\r \u00a0the\t\r \u00a0color\t\r \u00a0bar.\t\r \u00a0The\t\r \u00a0number\t\r \u00a0of\t\r \u00a0standard\t\r \u00a0deviations\t\r \u00a0from\t\r \u00a0the\t\r \u00a0mean\t\r \u00a0(log10\t\r \u00a0transformed)\t\r \u00a0is\t\r \u00a0shown\t\r \u00a0on\t\r \u00a0the\t\r \u00a0y-\u00ad\u2010axis\t\r \u00a0and\t\r \u00a0time\t\r \u00a0post\t\r \u00a0infection\t\r \u00a0is\t\r \u00a0shown\t\r \u00a0on\t\r \u00a0the\t\r \u00a0x-\u00ad\u2010axis.\t\r \u00a0B)\t\r \u00a0GO\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0regulated\t\r \u00a0phosphoproteins.\t\r \u00a0All\t\r \u00a0regulated\t\r \u00a0proteins\t\r \u00a0(containing\t\r \u00a0>\t\r \u00a0one\t\r \u00a0phosphopeptides\t\r \u00a0with\t\r \u00a0P<.01),\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0all\t\r \u00a0up-\u00ad\u2010regulated\t\r \u00a0proteins\t\r \u00a0(containing\t\r \u00a0>\t\r \u00a0one\t\r \u00a0phosphopeptides\t\r \u00a0with\t\r \u00a0membership\t\r \u00a0value\t\r \u00a0>\t\r \u00a00.6\t\r \u00a0from\t\r \u00a0up-\u00ad\u2010regulated\t\r \u00a0clusters\t\r \u00a0in\t\r \u00a0A)\t\r \u00a0and\t\r \u00a0down-\u00ad\u2010regulated\t\r \u00a0proteins\t\r \u00a0(containing\t\r \u00a0at\t\r \u00a0least\t\r \u00a0one\t\r \u00a0phosphopeptides\t\r \u00a0with\t\r \u00a0membership\t\r \u00a0value\t\r \u00a0>\t\r \u00a00.6\t\r \u00a0from\t\r \u00a0down-\u00ad\u2010regulated\t\r \u00a0clusters\t\r \u00a0in\t\r \u00a0A)\t\r \u00a0were\t\r \u00a0analyzed\t\r \u00a0for\t\r \u00a0enrichment\t\r \u00a0in\t\r \u00a0GO\t\r \u00a0terms.\t\r \u00a0GO\t\r \u00a0terms\t\r \u00a0for\t\r \u00a0biological\t\r \u00a0process\t\r \u00a0are\t\r \u00a0shown\t\r \u00a0on\t\r \u00a0the\t\r \u00a0x-\u00ad\u2010axis.\t\r \u00a0C)\t\r \u00a0Distribution\t\r \u00a0of\t\r \u00a0phosphorylation\t\r \u00a0dynamics\t\r \u00a0within\t\r \u00a0each\t\r \u00a0subcellular\t\r \u00a0fraction.\t\r \u00a0Down-\u00ad\u2010regulated\t\r \u00a0phosphosites\t\r \u00a0are\t\r \u00a0shown\t\r \u00a0in\t\r \u00a0red\t\r \u00a0and\t\r \u00a0up-\u00ad\u2010regulated\t\r \u00a0sites\t\r \u00a0are\t\r \u00a0shown\t\r \u00a0in\t\r \u00a0green.\t\r \u00a0Time\t\r \u00a0post\t\r \u00a0infection\t\r \u00a0is\t\r \u00a0shown\t\r \u00a0on\t\r \u00a0the\t\r \u00a0z-\u00ad\u2010axis.\t\r \u00a0\t\r \u00a0\t\r \u00a0 4.3.2\t\r \u00a0Validation\t\r \u00a0and\t\r \u00a0confirmation\t\r \u00a0of\t\r \u00a0phosphoproteomic\t\r \u00a0data\t\r \u00a0To\t\r \u00a0validate\t\r \u00a0the\t\r \u00a0phosphoproteomics\t\r \u00a0data,\t\r \u00a0profiles\t\r \u00a0for\t\r \u00a0several\t\r \u00a0phosphorylation\t\r \u00a0sites\t\r \u00a0were\t\r \u00a0confirmed\t\r \u00a0by\t\r \u00a0immunoblotting.\t\r \u00a0Phospho-\u00ad\u2010specific\t\r \u00a0antibodies\t\r \u00a0were\t\r \u00a0used\t\r \u00a0to\t\r \u00a0probe\t\r \u00a0phosphorylation\t\r \u00a0dynamics\t\r \u00a0within\t\r \u00a0nuclear,\t\r \u00a0membrane\t\r \u00a0and\t\r \u00a0cytosolic\t\r \u00a0fractions\t\r \u00a0either\t\r \u00a0mock\t\r \u00a0infected,\t\r \u00a0or\t\r \u00a0infected\t\r \u00a0for\t\r \u00a02,5,10\t\r \u00a0and\t\r \u00a020\t\r \u00a0min\t\r \u00a0with\t\r \u00a0WT\t\r \u00a0S.\t\r \u00a0Typhimurium.\t\r \u00a0As\t\r \u00a0seen\t\r \u00a0in\t\r \u00a0Figure\t\r \u00a04.3A,\t\r \u00a0these\t\r \u00a0profiles\t\r \u00a0compared\t\r \u00a0very\t\r \u00a0favorably\t\r \u00a0to\t\r \u00a0those\t\r \u00a0generated\t\r \u00a0by\t\r \u00a0quantitative\t\r \u00a0LC-\u00ad\u2010MSn.\t\r \u00a0The\t\r \u00a0SILAC\t\r \u00a0data\t\r \u00a0was\t\r \u00a0also\t\r \u00a0compared\t\r \u00a0to\t\r \u00a0all\t\r \u00a0protein\t\r \u00a0phosphorylation\t\r \u00a0signaling\t\r \u00a0events\t\r \u00a0previously\t\r \u00a0reported\t\r \u00a0in\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0during\t\r \u00a0Salmonella\t\r \u00a0invasion.\t\r \u00a0In\t\r \u00a0each\t\r \u00a0case\t\r \u00a0where\t\r \u00a0a\t\r \u00a0phosphosite\t\r \u00a0within\t\r \u00a0a\t\r \u00a0known\t\r \u00a0host\t\r \u00a0target\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0was\t\r \u00a0identified\t\r \u00a0in\t\r \u00a0the\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0data,\t\r \u00a0previously\t\r \u00a0reported\t\r \u00a0dynamics\t\r \u00a0were\t\r \u00a0confirmed\t\r \u00a0by\t\r \u00a0the\t\r \u00a0SILAC\t\r \u00a0profiles.\t\r \u00a0As\t\r \u00a0shown\t\r \u00a0in\t\r \u00a0Figures\t\r \u00a04.3A\t\r \u00a0and\t\r \u00a04.3B,\t\r \u00a0phosphorylation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0TxY\t\r \u00a0motif\t\r \u00a0in\t\r \u00a0ERK\t\r \u00a01\t\r \u00a0and\t\r \u00a02\t\r \u00a0increased\t\r \u00a0ten-\u00ad\u2010fold\t\r \u00a0during\t\r \u00a0Salmonella\t\r \u00a0infection,\t\r \u00a0which\t\r \u00a0is\t\r \u00a0consistent\t\r \u00a0with\t\r \u00a0previous\t\r \u00a0work\t\r \u00a0showing\t\r \u00a0activation\t\r \u00a0of\t\r \u00a0these\t\r \u00a0kinases\t\r \u00a0downstream\t\r \u00a0of\t\r \u00a0SopB\t\r \u00a0and\t\r \u00a0SopE\/E2(33,\t\r \u00a045).\t\r \u00a0Similarly,\t\r \u00a0up-\u00ad\u2010regulated\t\r \u00a0phosphorylations\t\r \u00a0of\t\r \u00a0threonine\t\r \u00a0pairs\t\r \u00a069\/71\t\r \u00a0and\t\r \u00a051\/53\t\r \u00a0were\t\r \u00a0observed\t\r \u00a0in\t\r \u00a0Cyclic\t\r \u00a0AMP-\u00ad\u2010dependent\t\r \u00a0transcription\t\r \u00a0factors\t\r \u00a0ATF2\t\r \u00a0and\t\r \u00a0ATF7,\t\r \u00a0which\t\r \u00a0are\t\r \u00a0components\t\r \u00a0of\t\r \u00a0the\t\r \u00a0AP-\u00ad\u20101\t\r \u00a0complex(511,\t\r \u00a0512).\t\r \u00a0Downstream\t\r \u00a0of\t\r \u00a0SopB,\t\r \u00a0we\t\r \u00a0observed\t\r \u00a0increased\t\r \u00a0phosphorylation\t\r \u00a0of\t\r \u00a0Ser-\u00ad\u2010474\t\r \u00a0from\t\r \u00a0Akt2,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0Ser-\u00ad\u2010588\t\r \u00a0of\t\r \u00a0an\t\r \u00a0 \t\r \u00a0\t\r \u00a0 119\t\r \u00a0 Akt\t\r \u00a0effector,\t\r \u00a0AS160\t\r \u00a0(Figure\t\r \u00a04.3b)(66,\t\r \u00a0461).\t\r \u00a0Phosphorylated\t\r \u00a0Ser-\u00ad\u2010181\t\r \u00a0in\t\r \u00a0PAK4\t\r \u00a0also\t\r \u00a0increased,\t\r \u00a0which\t\r \u00a0has\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0downstream\t\r \u00a0of\t\r \u00a0Akt\t\r \u00a0and\t\r \u00a0follows\t\r \u00a0activation\t\r \u00a0of\t\r \u00a0Cdc42\t\r \u00a0by\t\r \u00a0SopB\t\r \u00a0and\t\r \u00a0SopE\/E2(66,\t\r \u00a0513).\t\r \u00a0Regulation\t\r \u00a0at\t\r \u00a0Ser-\u00ad\u2010260\t\r \u00a0in\t\r \u00a0Serine\/threonine-\u00ad\u2010protein\t\r \u00a0kinase\t\r \u00a0A-\u00ad\u2010Raf\t\r \u00a0(ARaf)\t\r \u00a0was\t\r \u00a0also\t\r \u00a0expected,\t\r \u00a0as\t\r \u00a0SptP\t\r \u00a0is\t\r \u00a0known\t\r \u00a0to\t\r \u00a0regulate\t\r \u00a0signaling\t\r \u00a0through\t\r \u00a0the\t\r \u00a0ERK1\/2\t\r \u00a0pathway(178).\t\r \u00a0 Figure\t\r \u00a04.3\t\r \u00a0Validation\t\r \u00a0of\t\r \u00a0phosphoproteomics\t\r \u00a0data\t\r \u00a0 \t\r \u00a0 \t\r \u00a0\t\r \u00a0 120\t\r \u00a0 A)\t\r \u00a0Validation\t\r \u00a0of\t\r \u00a0SILAC\t\r \u00a0profiles\t\r \u00a0by\t\r \u00a0Western\t\r \u00a0blotting\t\r \u00a0with\t\r \u00a0phospho-\u00ad\u2010specific\t\r \u00a0antibodies.\t\r \u00a0B)\t\r \u00a0Phosphoproteomic\t\r \u00a0data\t\r \u00a0from\t\r \u00a0host\t\r \u00a0proteins\t\r \u00a0known\t\r \u00a0to\t\r \u00a0be\t\r \u00a0targeted\t\r \u00a0by\t\r \u00a0Salmonella.\t\r \u00a0Phosphosites\t\r \u00a0are\t\r \u00a0indicated\t\r \u00a0in\t\r \u00a0red\t\r \u00a0in\t\r \u00a0each\t\r \u00a0peptide\t\r \u00a0sequence.\t\r \u00a0In\t\r \u00a0each\t\r \u00a0case,\t\r \u00a0equal\t\r \u00a0protein\t\r \u00a0amounts\t\r \u00a0were\t\r \u00a0analyzed\t\r \u00a0from\t\r \u00a0each\t\r \u00a0time\t\r \u00a0point.\t\r \u00a0\t\r \u00a0\t\r \u00a0 4.3.3\t\r \u00a0Phosphorylation\t\r \u00a0dynamics\t\r \u00a0following\t\r \u00a0EGF\t\r \u00a0treatment\t\r \u00a0versus\t\r \u00a0Salmonella\t\r \u00a0 infection\t\r \u00a0Signaling\t\r \u00a0through\t\r \u00a0the\t\r \u00a0Epidermal\t\r \u00a0growth\t\r \u00a0factor\t\r \u00a0receptor\t\r \u00a0(EGFR)\t\r \u00a0represents\t\r \u00a0a\t\r \u00a0relatively\t\r \u00a0well-\u00ad\u2010characterized\t\r \u00a0pathway\t\r \u00a0initiated\t\r \u00a0by\t\r \u00a0extracellular\t\r \u00a0ligand-\u00ad\u2010receptor\t\r \u00a0binding,\t\r \u00a0and\t\r \u00a0proceeding\t\r \u00a0through\t\r \u00a0an\t\r \u00a0intracellular\t\r \u00a0MAPK\t\r \u00a0cascade\t\r \u00a0to\t\r \u00a0induce\t\r \u00a0transcription\t\r \u00a0of\t\r \u00a0genes\t\r \u00a0for\t\r \u00a0cell\t\r \u00a0growth\t\r \u00a0and\t\r \u00a0proliferation.\t\r \u00a0Conversely,\t\r \u00a0a\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0receptor\t\r \u00a0for\t\r \u00a0Salmonella\t\r \u00a0has\t\r \u00a0not\t\r \u00a0been\t\r \u00a0identified\t\r \u00a0and\t\r \u00a0no\t\r \u00a0known\t\r \u00a0signaling\t\r \u00a0cascades\t\r \u00a0are\t\r \u00a0induced\t\r \u00a0in\t\r \u00a0epithelial\t\r \u00a0cells\t\r \u00a0as\t\r \u00a0a\t\r \u00a0result\t\r \u00a0of\t\r \u00a0bacteria\t\r \u00a0binding\t\r \u00a0to\t\r \u00a0the\t\r \u00a0cell\t\r \u00a0surface(189).\t\r \u00a0Instead,\t\r \u00a0the\t\r \u00a0array\t\r \u00a0of\t\r \u00a0effector\t\r \u00a0proteins\t\r \u00a0injected\t\r \u00a0into\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0localize\t\r \u00a0to\t\r \u00a0various\t\r \u00a0compartments\t\r \u00a0within\t\r \u00a0the\t\r \u00a0host\t\r \u00a0cell,\t\r \u00a0in\t\r \u00a0some\t\r \u00a0cases\t\r \u00a0inducing\t\r \u00a0MAPK\t\r \u00a0signaling\t\r \u00a0for\t\r \u00a0cytokine\t\r \u00a0production\t\r \u00a0and\/or\t\r \u00a0stimulating\t\r \u00a0an\t\r \u00a0innate\t\r \u00a0immune\t\r \u00a0response.\t\r \u00a0Given\t\r \u00a0the\t\r \u00a0different\t\r \u00a0spatial\t\r \u00a0modes\t\r \u00a0of\t\r \u00a0action\t\r \u00a0then,\t\r \u00a0one\t\r \u00a0might\t\r \u00a0expect\t\r \u00a0that\t\r \u00a0signaling\t\r \u00a0downstream\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0would\t\r \u00a0not\t\r \u00a0necessarily\t\r \u00a0conform\t\r \u00a0to\t\r \u00a0that\t\r \u00a0of\t\r \u00a0canonical\t\r \u00a0signaling\t\r \u00a0pathways.\t\r \u00a0Olsen\t\r \u00a0et\t\r \u00a0al.\t\r \u00a0have\t\r \u00a0previously\t\r \u00a0reported\t\r \u00a0the\t\r \u00a0averaged\t\r \u00a0temporal\t\r \u00a0profiles\t\r \u00a0of\t\r \u00a0all\t\r \u00a0up-\u00ad\u2010regulated\t\r \u00a0pY,\t\r \u00a0pS\t\r \u00a0and\t\r \u00a0pT\t\r \u00a0sites\t\r \u00a0detected\t\r \u00a0downstream\t\r \u00a0of\t\r \u00a0EGF\t\r \u00a0stimulation\t\r \u00a0and\t\r \u00a0observed\t\r \u00a0that\t\r \u00a0pY\t\r \u00a0regulation\t\r \u00a0occurs\t\r \u00a0much\t\r \u00a0earlier\t\r \u00a0than\t\r \u00a0pS\t\r \u00a0or\t\r \u00a0pT\t\r \u00a0regulation\t\r \u00a0(Figure\t\r \u00a04.4A)(297).\t\r \u00a0Treating\t\r \u00a0our\t\r \u00a0data\t\r \u00a0similarly,\t\r \u00a0we\t\r \u00a0observed\t\r \u00a0similar\t\r \u00a0rates\t\r \u00a0for\t\r \u00a0all\t\r \u00a0three\t\r \u00a0sites\t\r \u00a0indicating\t\r \u00a0that,\t\r \u00a0unlike\t\r \u00a0EGF,\t\r \u00a0Salmonella\t\r \u00a0does\t\r \u00a0not\t\r \u00a0trigger\t\r \u00a0rapid\t\r \u00a0changes\t\r \u00a0in\t\r \u00a0pY\t\r \u00a0at\t\r \u00a0the\t\r \u00a0cell\t\r \u00a0membrane\t\r \u00a0(Figure\t\r \u00a04.4A).\t\r \u00a0It\t\r \u00a0is\t\r \u00a0noteworthy\t\r \u00a0however,\t\r \u00a0that\t\r \u00a0the\t\r \u00a0magnitude\t\r \u00a0of\t\r \u00a0pY\t\r \u00a0 \t\r \u00a0\t\r \u00a0 121\t\r \u00a0 regulation\t\r \u00a0by\t\r \u00a0Salmonella\t\r \u00a0is\t\r \u00a0much\t\r \u00a0higher\t\r \u00a0than\t\r \u00a0that\t\r \u00a0for\t\r \u00a0pS\t\r \u00a0and\t\r \u00a0pT.\t\r \u00a0While\t\r \u00a0we\t\r \u00a0would\t\r \u00a0not\t\r \u00a0necessarily\t\r \u00a0expect\t\r \u00a0a\t\r \u00a0great\t\r \u00a0degree\t\r \u00a0of\t\r \u00a0overlap\t\r \u00a0between\t\r \u00a0phosphorylation\t\r \u00a0sites\t\r \u00a0regulated\t\r \u00a0by\t\r \u00a0Salmonella\t\r \u00a0and\t\r \u00a0EGF,\t\r \u00a0a\t\r \u00a0more\t\r \u00a0detailed\t\r \u00a0comparison\t\r \u00a0of\t\r \u00a0individual\t\r \u00a0sites\t\r \u00a0is\t\r \u00a0illuminating.\t\r \u00a0Seventy-\u00ad\u2010two\t\r \u00a0regulated\t\r \u00a0phosphopeptides\t\r \u00a0were\t\r \u00a0common\t\r \u00a0between\t\r \u00a0the\t\r \u00a0two\t\r \u00a0datasets,\t\r \u00a0with\t\r \u00a0only\t\r \u00a0half\t\r \u00a0of\t\r \u00a0them\t\r \u00a0showing\t\r \u00a0vaguely\t\r \u00a0similar\t\r \u00a0patterns\t\r \u00a0of\t\r \u00a0regulation\t\r \u00a0(Figure\t\r \u00a04.4B).\t\r \u00a0This\t\r \u00a0reveals\t\r \u00a0that\t\r \u00a0even\t\r \u00a0individual\t\r \u00a0proteins\t\r \u00a0can\t\r \u00a0play\t\r \u00a0very\t\r \u00a0different\t\r \u00a0roles\t\r \u00a0in\t\r \u00a0signaling\t\r \u00a0pathways.\t\r \u00a0In\t\r \u00a0addition,\t\r \u00a0activation\t\r \u00a0of\t\r \u00a0ERK2\t\r \u00a0was\t\r \u00a0delayed\t\r \u00a0during\t\r \u00a0 Salmonella\t\r \u00a0as\t\r \u00a0compared\t\r \u00a0to\t\r \u00a0EGF\t\r \u00a0stimulation,\t\r \u00a0indicating\t\r \u00a0that\t\r \u00a0stimulation\t\r \u00a0by\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0delivered\t\r \u00a0effectors\t\r \u00a0lags\t\r \u00a0that\t\r \u00a0of\t\r \u00a0receptor\t\r \u00a0activation\t\r \u00a0at\t\r \u00a0the\t\r \u00a0cell\t\r \u00a0membrane.\t\r \u00a0On\t\r \u00a0a\t\r \u00a0larger\t\r \u00a0scale,\t\r \u00a0are\t\r \u00a0there\t\r \u00a0specific\t\r \u00a0functional\t\r \u00a0classes\t\r \u00a0of\t\r \u00a0proteins\t\r \u00a0that\t\r \u00a0are\t\r \u00a0used\t\r \u00a0in\t\r \u00a0this\t\r \u00a0way?\t\r \u00a0GO\t\r \u00a0terms\t\r \u00a0for\t\r \u00a0cellular\t\r \u00a0compartment\t\r \u00a0were\t\r \u00a0very\t\r \u00a0similar\t\r \u00a0following\t\r \u00a0EGF\t\r \u00a0and\t\r \u00a0Salmonella\t\r \u00a0treatment,\t\r \u00a0but\t\r \u00a0terms\t\r \u00a0for\t\r \u00a0biological\t\r \u00a0processes\t\r \u00a0relating\t\r \u00a0to\t\r \u00a0transcriptional\t\r \u00a0regulation\t\r \u00a0were\t\r \u00a0significantly\t\r \u00a0enriched\t\r \u00a0by\t\r \u00a0EGF\t\r \u00a0stimulation,\t\r \u00a0while\t\r \u00a0those\t\r \u00a0relating\t\r \u00a0to\t\r \u00a0small\t\r \u00a0GTPase\t\r \u00a0mediated\t\r \u00a0signaling\t\r \u00a0transduction\t\r \u00a0and\t\r \u00a0apoptosis\t\r \u00a0were\t\r \u00a0strikingly\t\r \u00a0over-\u00ad\u2010represented\t\r \u00a0during\t\r \u00a0Salmonella\t\r \u00a0infection\t\r \u00a0(Figure\t\r \u00a04.4C).\t\r \u00a0\t\r \u00a0 \t\r \u00a0\t\r \u00a0 122\t\r \u00a0 Figure\t\r \u00a04.4\t\r \u00a0Comparison\t\r \u00a0of\t\r \u00a0EGF-\u00ad\u2010\t\r \u00a0and\t\r \u00a0Salmonella-\u00ad\u2010induced\t\r \u00a0signaling\t\r \u00a0 \t\r \u00a0 \t\r \u00a0\t\r \u00a0 123\t\r \u00a0 A)\t\r \u00a0Average\t\r \u00a0SILAC\t\r \u00a0profiles\t\r \u00a0for\t\r \u00a0all\t\r \u00a0up-\u00ad\u2010regulated\t\r \u00a0phospho-\u00ad\u2010serine,\t\r \u00a0-\u00ad\u2010threonine,\t\r \u00a0and\t\r \u00a0\u2013tyrosine\t\r \u00a0residues.\t\r \u00a0Dashed\t\r \u00a0lines\t\r \u00a0represent\t\r \u00a0data\t\r \u00a0from\t\r \u00a0EGF\t\r \u00a0stimulation\t\r \u00a0and\t\r \u00a0solid\t\r \u00a0lines\t\r \u00a0represent\t\r \u00a0data\t\r \u00a0from\t\r \u00a0Salmonella\t\r \u00a0infection.\t\r \u00a0B)\t\r \u00a0Hierarchical\t\r \u00a0clustering\t\r \u00a0of\t\r \u00a0all\t\r \u00a0dynamic\t\r \u00a0phosphopeptides\t\r \u00a0similar\t\r \u00a0between\t\r \u00a0the\t\r \u00a0EGF\t\r \u00a0and\t\r \u00a0 Salmonella\t\r \u00a0data.\t\r \u00a0Time\t\r \u00a0post\t\r \u00a0EGF\t\r \u00a0and\t\r \u00a0Salmonella\t\r \u00a0treatment\t\r \u00a0is\t\r \u00a0indicated,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0a\t\r \u00a0color\t\r \u00a0scale\t\r \u00a0showing\t\r \u00a0log10\t\r \u00a0fold\t\r \u00a0change\t\r \u00a0within\t\r \u00a0each\t\r \u00a0profile.\t\r \u00a0Profiles\t\r \u00a0corresponding\t\r \u00a0to\t\r \u00a0the\t\r \u00a0peptide\t\r \u00a0VADPDHDHTGFLpTEpYATR\t\r \u00a0from\t\r \u00a0ERK2\t\r \u00a0are\t\r \u00a0indicated\t\r \u00a0by\t\r \u00a0an\t\r \u00a0arrow.\t\r \u00a0C)\t\r \u00a0GO\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0up-\u00ad\u2010regulated\t\r \u00a0phosphoproteins\t\r \u00a0following\t\r \u00a0EGF\t\r \u00a0and\t\r \u00a0Salmonella\t\r \u00a0stimulation.\t\r \u00a0All\t\r \u00a0phosphoproteins\t\r \u00a0up-\u00ad\u2010regulated\t\r \u00a0during\t\r \u00a0 Salmonella\t\r \u00a0infection\t\r \u00a0(refer\t\r \u00a0to\t\r \u00a0Figure\t\r \u00a04.2B),\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0a\t\r \u00a0similar\t\r \u00a0set\t\r \u00a0following\t\r \u00a0EGF\t\r \u00a0treatment\t\r \u00a0were\t\r \u00a0analyzed\t\r \u00a0for\t\r \u00a0enrichment\t\r \u00a0in\t\r \u00a0GO\t\r \u00a0terms.\t\r \u00a0GO\t\r \u00a0terms\t\r \u00a0for\t\r \u00a0biological\t\r \u00a0process\t\r \u00a0are\t\r \u00a0shown\t\r \u00a0on\t\r \u00a0the\t\r \u00a0x-\u00ad\u2010axis.\t\r \u00a0\t\r \u00a0\t\r \u00a0 4.3.4\t\r \u00a0Salmonella\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0protein\t\r \u00a0SipB\t\r \u00a0is\t\r \u00a0phosphorylated\t\r \u00a0in\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0The\t\r \u00a0approach\t\r \u00a0depicted\t\r \u00a0in\t\r \u00a0Figure\t\r \u00a04.1\t\r \u00a0was\t\r \u00a0designed\t\r \u00a0to\t\r \u00a0identify\t\r \u00a0phosphorylation\t\r \u00a0events\t\r \u00a0in\t\r \u00a0host\t\r \u00a0cells,\t\r \u00a0but\t\r \u00a0of\t\r \u00a0course\t\r \u00a0bacterial\t\r \u00a0phosphopeptides\t\r \u00a0could\t\r \u00a0also\t\r \u00a0be\t\r \u00a0present,\t\r \u00a0albeit\t\r \u00a0lacking\t\r \u00a0a\t\r \u00a0SILAC\t\r \u00a0label\t\r \u00a0for\t\r \u00a0quantitation.\t\r \u00a0To\t\r \u00a0identify\t\r \u00a0potential\t\r \u00a0phosphorylation\t\r \u00a0sites\t\r \u00a0in\t\r \u00a0Salmonella\t\r \u00a0proteins\t\r \u00a0after\t\r \u00a0infection,\t\r \u00a0the\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0data\t\r \u00a0was\t\r \u00a0separately\t\r \u00a0searched\t\r \u00a0against\t\r \u00a0a\t\r \u00a0database\t\r \u00a0containing\t\r \u00a0all\t\r \u00a0predicted\t\r \u00a0peptides\t\r \u00a0from\t\r \u00a0S.\t\r \u00a0Typhimurium\t\r \u00a0strain\t\r \u00a0SL1344.\t\r \u00a0Interestingly,\t\r \u00a0a\t\r \u00a0triply\t\r \u00a0phosphorylated\t\r \u00a0peptide\t\r \u00a0from\t\r \u00a0the\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0encoded\t\r \u00a0effector\t\r \u00a0SipB\t\r \u00a0was\t\r \u00a0identified\t\r \u00a0(IMGCIGKVLGALL(pT)IV(pS)VVAAVF(pT)GGA(pS)LALAAVGLAVMVADEIVK;\t\r \u00a0three\t\r \u00a0phosphorylations\t\r \u00a0were\t\r \u00a0detected\t\r \u00a0but\t\r \u00a0the\t\r \u00a0spectrum\t\r \u00a0does\t\r \u00a0not\t\r \u00a0allow\t\r \u00a0unambiguous\t\r \u00a0assignment\t\r \u00a0of\t\r \u00a0the\t\r \u00a0specific\t\r \u00a0sites).\t\r \u00a0SipB\t\r \u00a0is\t\r \u00a0one\t\r \u00a0of\t\r \u00a0three\t\r \u00a0translocon\t\r \u00a0proteins\t\r \u00a0secreted\t\r \u00a0by\t\r \u00a0T3SS-\u00ad\u20101,\t\r \u00a0and\t\r \u00a0is\t\r \u00a0required\t\r \u00a0for\t\r \u00a0intimate\t\r \u00a0attachment\t\r \u00a0to\t\r \u00a0host\t\r \u00a0cells,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0delivery\t\r \u00a0of\t\r \u00a0bacterial\t\r \u00a0effectors(133,\t\r \u00a0135).\t\r \u00a0The\t\r \u00a0peptide\t\r \u00a0falls\t\r \u00a0within\t\r \u00a0a\t\r \u00a0central\t\r \u00a0region\t\r \u00a0of\t\r \u00a0SipB\t\r \u00a0that\t\r \u00a0is\t\r \u00a0required\t\r \u00a0for\t\r \u00a0its\t\r \u00a0secretion(133).\t\r \u00a0This\t\r \u00a0indicates\t\r \u00a0that\t\r \u00a0phosphorylation\t\r \u00a0may\t\r \u00a0regulate\t\r \u00a0its\t\r \u00a0insertion\t\r \u00a0and\/or\t\r \u00a0delivery\t\r \u00a0to\t\r \u00a0T3SS-\u00ad\u20101,\t\r \u00a0potentially\t\r \u00a0through\t\r \u00a0binding\t\r \u00a0forkhead-\u00ad\u2010associated\t\r \u00a0domains\t\r \u00a0(which\t\r \u00a0bind\t\r \u00a0pS\t\r \u00a0and\t\r \u00a0pT)\t\r \u00a0located\t\r \u00a0on\t\r \u00a0other\t\r \u00a0T3SS\t\r \u00a0components(514).\t\r \u00a0\t\r \u00a0 \t\r \u00a0\t\r \u00a0 124\t\r \u00a0 4.3.5\t\r \u00a0Novel\t\r \u00a0host\t\r \u00a0phosphorylation-\u00ad\u2010sites\t\r \u00a0regulated\t\r \u00a0during\t\r \u00a0Salmonella\t\r \u00a0infection\t\r \u00a0A\t\r \u00a0main\t\r \u00a0trigger\t\r \u00a0of\t\r \u00a0innate\t\r \u00a0immune\t\r \u00a0responses\t\r \u00a0is\t\r \u00a0the\t\r \u00a0recognition\t\r \u00a0of\t\r \u00a0pathogen-\u00ad\u2010associated\t\r \u00a0molecular\t\r \u00a0patterns\t\r \u00a0(PAMPs)\t\r \u00a0by\t\r \u00a0Toll-\u00ad\u2010like\t\r \u00a0receptors\t\r \u00a0(TLRs)\t\r \u00a0and\t\r \u00a0NLRs,\t\r \u00a0leading\t\r \u00a0to\t\r \u00a0MAPK\t\r \u00a0signaling\t\r \u00a0and\t\r \u00a0the\t\r \u00a0production\t\r \u00a0of\t\r \u00a0pro-\u00ad\u2010inflammatory\t\r \u00a0cytokines.\t\r \u00a0Professional\t\r \u00a0phagocytes\t\r \u00a0express\t\r \u00a0such\t\r \u00a0proteins\t\r \u00a0widely\t\r \u00a0but\t\r \u00a0epithelial\t\r \u00a0cells,\t\r \u00a0which\t\r \u00a0are\t\r \u00a0continually\t\r \u00a0exposed\t\r \u00a0to\t\r \u00a0large\t\r \u00a0numbers\t\r \u00a0of\t\r \u00a0bacteria,\t\r \u00a0down-\u00ad\u2010regulate\t\r \u00a0signaling\t\r \u00a0through\t\r \u00a0these\t\r \u00a0receptors\t\r \u00a0to\t\r \u00a0prevent\t\r \u00a0uncontrolled\t\r \u00a0inflammation(515).\t\r \u00a0Remarkably,\t\r \u00a0 Salmonella\t\r \u00a0actively\t\r \u00a0stimulate\t\r \u00a0innate\t\r \u00a0immune\t\r \u00a0responses\t\r \u00a0in\t\r \u00a0epithelial\t\r \u00a0cells\t\r \u00a0through\t\r \u00a0mechanisms\t\r \u00a0dependent\t\r \u00a0on\t\r \u00a0effector\t\r \u00a0proteins\t\r \u00a0SopE\/E2\t\r \u00a0and\t\r \u00a0SopB,\t\r \u00a0but\t\r \u00a0which\t\r \u00a0do\t\r \u00a0not\t\r \u00a0involve\t\r \u00a0TLRs\t\r \u00a0or\t\r \u00a0NLRs(189).\t\r \u00a0Salmonella\t\r \u00a0use\t\r \u00a0tetrathionate\t\r \u00a0derived\t\r \u00a0from\t\r \u00a0reactive\t\r \u00a0oxygen\t\r \u00a0species\t\r \u00a0generated\t\r \u00a0during\t\r \u00a0inflammation\t\r \u00a0as\t\r \u00a0an\t\r \u00a0electron\t\r \u00a0acceptor\t\r \u00a0for\t\r \u00a0respiration(191).\t\r \u00a0This\t\r \u00a0gives\t\r \u00a0them\t\r \u00a0a\t\r \u00a0growth\t\r \u00a0advantage\t\r \u00a0over\t\r \u00a0commensal\t\r \u00a0microbes,\t\r \u00a0and\t\r \u00a0inflammation\t\r \u00a0has\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0enhance\t\r \u00a0transmission\t\r \u00a0and\t\r \u00a0growth\t\r \u00a0of\t\r \u00a0 Salmonella\t\r \u00a0in\t\r \u00a0the\t\r \u00a0intestinal\t\r \u00a0lumen(190).\t\r \u00a0Thus,\t\r \u00a0host\t\r \u00a0signaling\t\r \u00a0events\t\r \u00a0observed\t\r \u00a0during\t\r \u00a0infection\t\r \u00a0likely\t\r \u00a0represent\t\r \u00a0novel\t\r \u00a0components\t\r \u00a0of\t\r \u00a0these\t\r \u00a0pathways\t\r \u00a0that\t\r \u00a0may\t\r \u00a0be\t\r \u00a0central\t\r \u00a0to\t\r \u00a0pathogenesis\t\r \u00a0and\t\r \u00a0disease.\t\r \u00a0Within\t\r \u00a0the\t\r \u00a0WT\t\r \u00a0dataset,\t\r \u00a0several\t\r \u00a0dynamic\t\r \u00a0phosphosites\t\r \u00a0likely\t\r \u00a0represent\t\r \u00a0novel\t\r \u00a0host\t\r \u00a0targets\t\r \u00a0of\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0effectors\t\r \u00a0(Table\t\r \u00a04.1).\t\r \u00a0For\t\r \u00a0example,\t\r \u00a0up-\u00ad\u2010regulation\t\r \u00a0of\t\r \u00a0activating\t\r \u00a0sites\t\r \u00a0in\t\r \u00a0IQGAP1\t\r \u00a0and\t\r \u00a0Protein-\u00ad\u2010serine\/threonine\t\r \u00a0kinase\t\r \u00a0D3\t\r \u00a0(PRKD3)\t\r \u00a0were\t\r \u00a0observed.\t\r \u00a0As\t\r \u00a0both\t\r \u00a0of\t\r \u00a0these\t\r \u00a0are\t\r \u00a0PKC\t\r \u00a0substrates,\t\r \u00a0these\t\r \u00a0likely\t\r \u00a0represent\t\r \u00a0novel\t\r \u00a0signaling\t\r \u00a0targets\t\r \u00a0downstream\t\r \u00a0of\t\r \u00a0SipA-\u00ad\u2010dependent\t\r \u00a0PKC\t\r \u00a0activation(147,\t\r \u00a0164,\t\r \u00a0516,\t\r \u00a0517).\t\r \u00a0Similarly,\t\r \u00a0dynamic\t\r \u00a0sites\t\r \u00a0in\t\r \u00a0Histone\t\r \u00a0deacetylase\t\r \u00a01\t\r \u00a0(HDAC1)\t\r \u00a0and\t\r \u00a0Non-\u00ad\u2010histone\t\r \u00a0chromosomal\t\r \u00a0protein\t\r \u00a0HMG-\u00ad\u201014\t\r \u00a0(HMG14),\t\r \u00a0both\t\r \u00a0of\t\r \u00a0which\t\r \u00a0are\t\r \u00a0transcriptional\t\r \u00a0regulators\t\r \u00a0 \t\r \u00a0\t\r \u00a0 125\t\r \u00a0 phosphorylated\t\r \u00a0by\t\r \u00a0casein\t\r \u00a0kinases\t\r \u00a01\t\r \u00a0and\t\r \u00a02,\t\r \u00a0implicates\t\r \u00a0the\t\r \u00a0possible\t\r \u00a0involvement\t\r \u00a0of\t\r \u00a0these\t\r \u00a0kinases\t\r \u00a0as\t\r \u00a0well(518,\t\r \u00a0519).\t\r \u00a0 Table\t\r \u00a04.1\t\r \u00a0Selected\t\r \u00a0novel\t\r \u00a0host\t\r \u00a0phosphosites\t\r \u00a0regulated\t\r \u00a0during\t\r \u00a0Salmonella\t\r \u00a0infection\t\r \u00a0 Gene\t\r \u00a0 Name\t\r \u00a0 Phosphosite\t\r \u00a0Regulation*\t\r \u00a0 Protein\t\r \u00a0 Function\t\r \u00a0 Phosphosite\t\r \u00a0 Function\t\r \u00a0 Kinase\/Phosphatase\t\r \u00a0 Reference\t\r \u00a0BAD\t\r \u00a0 S99\t\r \u00a0 0.34+\/-\u00ad\u20100.00\t\r \u00a0 promotes\t\r \u00a0apoptosis\t\r \u00a0 Inhibits\t\r \u00a0pro-\u00ad\u2010apoptotic\t\r \u00a0function\t\r \u00a0 Akt\t\r \u00a0 (520,\t\r \u00a0521)\t\r \u00a0CFL1\t\r \u00a0 S3\t\r \u00a0 -\u00ad\u20100.30+\/-\u00ad\u20100.06\t\r \u00a0 actin\t\r \u00a0 Inhibits\t\r \u00a0actin\t\r \u00a0binding\t\r \u00a0 LIMK\t\r \u00a0 (522,\t\r \u00a0523)\t\r \u00a0CTNNB1\t\r \u00a0 S675\t\r \u00a0 0.30+\/-\u00ad\u20100.04\t\r \u00a0 cell\t\r \u00a0adhesion\t\r \u00a0and\t\r \u00a0Wnt\t\r \u00a0signaling\t\r \u00a0 Prevents\t\r \u00a0ubiquitylation\t\r \u00a0 PKA\t\r \u00a0 (524)\t\r \u00a0HDAC1\t\r \u00a0 S421,\t\r \u00a0S423\t\r \u00a0 0.20\t\r \u00a0 inhibits\t\r \u00a0transcription\t\r \u00a0 activates\t\r \u00a0deacetylation\t\r \u00a0activity\t\r \u00a0 CK1\t\r \u00a0 (518)\t\r \u00a0HMG14\t\r \u00a0 S7\t\r \u00a0 0.78+\/-\u00ad\u20100.04\t\r \u00a0 regulates\t\r \u00a0transcription\t\r \u00a0 unknown\t\r \u00a0 CK2\t\r \u00a0 (519)\t\r \u00a0IQGAP1\t\r \u00a0 S1443\t\r \u00a0 0.42+\/-\u00ad\u20100.05\t\r \u00a0 scaffold\t\r \u00a0 induces\t\r \u00a0interaction\t\r \u00a0 PKC\t\r \u00a0 (516)\t\r \u00a0MSK2\t\r \u00a0 S343,\t\r \u00a0S347\t\r \u00a0 0.91+\/-\u00ad\u20100.18\t\r \u00a0 kinase\t\r \u00a0 kinase\t\r \u00a0activation\t\r \u00a0 P38\t\r \u00a0 (525)\t\r \u00a0PRAS40\t\r \u00a0 S221\t\r \u00a0 -\u00ad\u20100.45+\/-\u00ad\u20100.20\t\r \u00a0 subunit\t\r \u00a0of\t\r \u00a0mTORC1\t\r \u00a0 relieves\t\r \u00a0mTOR\t\r \u00a0inhibition\t\r \u00a0 mTORC1\t\r \u00a0 (526)\t\r \u00a0PRKD3\t\r \u00a0 S735\t\r \u00a0 0.25+\/-\u00ad\u20100.09\t\r \u00a0 prolonged\t\r \u00a0DAG\t\r \u00a0signaling\t\r \u00a0 kinase\t\r \u00a0activation\t\r \u00a0 PRKD3\t\r \u00a0 (517)\t\r \u00a0RAC1\t\r \u00a0 S71\t\r \u00a0 0.64\t\r \u00a0 GTPase\t\r \u00a0 inhibits\t\r \u00a0GTP\t\r \u00a0binding\t\r \u00a0 Akt\t\r \u00a0 (527)\t\r \u00a0RPS6\t\r \u00a0 S240\t\r \u00a0 0.73+\/-\u00ad\u20100.30\t\r \u00a0 cell\t\r \u00a0growth\t\r \u00a0and\t\r \u00a0proliferation\t\r \u00a0 may\t\r \u00a0promote\t\r \u00a0mRNA\t\r \u00a0binding\t\r \u00a0 S6K,\t\r \u00a0Akt,\t\r \u00a0RSK1\t\r \u00a0 (528,\t\r \u00a0529)\t\r \u00a0RSK1\t\r \u00a0 S368,\t\r \u00a0S389\t\r \u00a0 0.66,\t\r \u00a00.84\t\r \u00a0 Cell\t\r \u00a0growth\t\r \u00a0through\t\r \u00a0CREB\t\r \u00a0activation\t\r \u00a0 Kinase\t\r \u00a0activation\t\r \u00a0 ERK1\/2\t\r \u00a0 (530)\t\r \u00a0 SHC1\t\r \u00a0 S139\t\r \u00a0 0.35+\/-\u00ad\u20100.05\t\r \u00a0 signaling\t\r \u00a0adaptor\t\r \u00a0 down-\u00ad\u2010regulation\t\r \u00a0 unknown\t\r \u00a0 (531)\t\r \u00a0SRC1\t\r \u00a0 Y419\t\r \u00a0 0.34\t\r \u00a0 kinase\t\r \u00a0 activates\t\r \u00a0kinase\t\r \u00a0 SRC1\t\r \u00a0 (532)\t\r \u00a0*\t\r \u00a0Values\t\r 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\u00a0more\t\r \u00a0than\t\r \u00a0two-\u00ad\u2010fold\t\r \u00a0during\t\r \u00a0infection\t\r \u00a0with\t\r \u00a0WT\t\r \u00a0S.\t\r \u00a0Typhimurium\t\r \u00a0were\t\r \u00a0scored\t\r \u00a0against\t\r \u00a0the\t\r \u00a0substrate\t\r \u00a0specificity\t\r \u00a0determinants\t\r \u00a0for\t\r \u00a0493\t\r \u00a0human\t\r \u00a0kinases.\t\r \u00a0Interestingly,\t\r \u00a0Akt\t\r \u00a0and\t\r \u00a0PKC\t\r \u00a0(known\t\r \u00a0targets\t\r \u00a0of\t\r \u00a0SopB\t\r \u00a0and\t\r \u00a0SipA)\t\r \u00a0were\t\r \u00a0well\t\r \u00a0represented\t\r \u00a0among\t\r \u00a0the\t\r \u00a0top\t\r \u00a020\t\r \u00a0scoring\t\r \u00a0kinases,\t\r \u00a0as\t\r \u00a0were\t\r \u00a0p70S6K,\t\r \u00a0mTOR,\t\r \u00a0and\t\r \u00a0the\t\r \u00a0PIM\t\r \u00a0family\t\r \u00a0kinases\t\r \u00a0(Table\t\r \u00a04.2).\t\r \u00a0In\t\r \u00a0addition,\t\r \u00a0among\t\r \u00a0the\t\r \u00a0total\t\r \u00a0241\t\r \u00a0phosphosites\t\r \u00a0identified\t\r \u00a0from\t\r \u00a066\t\r 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\u00a0 10\t\r \u00a0 5\t\r \u00a0 6\t\r \u00a0 8\t\r \u00a0 5\t\r \u00a0 6\t\r \u00a0 1\t\r \u00a0 5\t\r \u00a0 4\t\r \u00a0 4\t\r \u00a0 1\t\r \u00a0 2\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 3\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 104\t\r \u00a0PIM3\t\r \u00a0 6\t\r \u00a0 20\t\r \u00a0 13\t\r \u00a0 6\t\r \u00a0 6\t\r \u00a0 5\t\r \u00a0 6\t\r \u00a0 3\t\r \u00a0 5\t\r \u00a0 4\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 1\t\r \u00a0 1\t\r \u00a0 2\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 4\t\r \u00a0 3\t\r \u00a0 5\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 100\t\r \u00a0PIM2\t\r \u00a0 4\t\r \u00a0 8\t\r \u00a0 13\t\r \u00a0 7\t\r \u00a0 7\t\r \u00a0 7\t\r \u00a0 6\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 5\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 6\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 1\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 2\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 1\t\r \u00a0 2\t\r \u00a0 82\t\r \u00a0PKC\u03b1\t\r \u00a0 5\t\r \u00a0 11\t\r \u00a0 6\t\r \u00a0 3\t\r \u00a0 4\t\r \u00a0 6\t\r \u00a0 3\t\r \u00a0 4\t\r \u00a0 4\t\r \u00a0 1\t\r \u00a0 4\t\r \u00a0 6\t\r \u00a0 1\t\r \u00a0 3\t\r \u00a0 2\t\r \u00a0 0\t\r \u00a0 1\t\r \u00a0 3\t\r \u00a0 1\t\r \u00a0 4\t\r \u00a0 0\t\r \u00a0 3\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 1\t\r \u00a0 79\t\r \u00a0PKC\u03b2\t\r \u00a0 12\t\r \u00a0 9\t\r \u00a0 3\t\r \u00a0 2\t\r \u00a0 5\t\r \u00a0 5\t\r \u00a0 3\t\r \u00a0 4\t\r \u00a0 0\t\r \u00a0 2\t\r \u00a0 7\t\r \u00a0 3\t\r \u00a0 5\t\r \u00a0 3\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 4\t\r \u00a0 1\t\r \u00a0 1\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 1\t\r \u00a0 77\t\r \u00a0PKG1\t\r \u00a0 4\t\r \u00a0 2\t\r \u00a0 8\t\r \u00a0 6\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 5\t\r \u00a0 4\t\r \u00a0 4\t\r \u00a0 0\t\r \u00a0 1\t\r \u00a0 2\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 2\t\r \u00a0 4\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 5\t\r \u00a0 4\t\r \u00a0 0\t\r \u00a0 7\t\r \u00a0 6\t\r \u00a0 2\t\r \u00a0 2\t\r \u00a0 77\t\r \u00a0VACAMKL\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 2\t\r \u00a0 4\t\r \u00a0 4\t\r \u00a0 2\t\r \u00a0 6\t\r \u00a0 5\t\r \u00a0 10\t\r \u00a0 2\t\r \u00a0 4\t\r \u00a0 4\t\r \u00a0 7\t\r \u00a0 3\t\r \u00a0 4\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 2\t\r \u00a0 2\t\r \u00a0 0\t\r \u00a0 2\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 69\t\r \u00a0p70S6K\t\r \u00a0 4\t\r \u00a0 8\t\r \u00a0 4\t\r \u00a0 5\t\r \u00a0 11\t\r \u00a0 3\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 2\t\r \u00a0 4\t\r \u00a0 4\t\r \u00a0 0\t\r \u00a0 2\t\r \u00a0 0\t\r \u00a0 3\t\r \u00a0 3\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 3\t\r \u00a0 0\t\r \u00a0 4\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 2\t\r \u00a0 68\t\r \u00a0mTOR\t\r \u00a0 32\t\r \u00a0 5\t\r \u00a0 3\t\r \u00a0 5\t\r \u00a0 4\t\r \u00a0 4\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 2\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 1\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 66\t\r \u00a0PKC\u03b5\t\r \u00a0 4\t\r \u00a0 1\t\r \u00a0 2\t\r \u00a0 2\t\r \u00a0 6\t\r \u00a0 2\t\r \u00a0 4\t\r \u00a0 2\t\r \u00a0 3\t\r \u00a0 8\t\r \u00a0 3\t\r \u00a0 7\t\r \u00a0 2\t\r \u00a0 0\t\r \u00a0 3\t\r \u00a0 3\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 1\t\r \u00a0 1\t\r \u00a0 1\t\r \u00a0 3\t\r \u00a0 3\t\r \u00a0 1\t\r \u00a0 65\t\r \u00a0SGK2\t\r \u00a0 1\t\r \u00a0 1\t\r \u00a0 1\t\r \u00a0 5\t\r \u00a0 6\t\r \u00a0 11\t\r \u00a0 7\t\r \u00a0 3\t\r \u00a0 5\t\r \u00a0 3\t\r \u00a0 2\t\r \u00a0 3\t\r \u00a0 3\t\r \u00a0 2\t\r \u00a0 2\t\r \u00a0 3\t\r \u00a0 1\t\r \u00a0 1\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 63\t\r \u00a0AKT2\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 2\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 5\t\r \u00a0 4\t\r \u00a0 5\t\r \u00a0 5\t\r \u00a0 2\t\r \u00a0 4\t\r \u00a0 3\t\r \u00a0 5\t\r \u00a0 3\t\r \u00a0 4\t\r \u00a0 3\t\r \u00a0 3\t\r \u00a0 4\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 2\t\r \u00a0 0\t\r \u00a0 4\t\r \u00a0 59\t\r \u00a0AKT3\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 2\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 3\t\r \u00a0 3\t\r \u00a0 6\t\r \u00a0 3\t\r \u00a0 7\t\r \u00a0 3\t\r \u00a0 3\t\r \u00a0 3\t\r \u00a0 3\t\r \u00a0 3\t\r \u00a0 2\t\r \u00a0 2\t\r \u00a0 4\t\r \u00a0 2\t\r \u00a0 2\t\r \u00a0 0\t\r \u00a0 4\t\r \u00a0 4\t\r \u00a0 59\t\r \u00a0PKC\u03c2\t\r \u00a0 2\t\r \u00a0 0\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 1\t\r \u00a0 3\t\r \u00a0 3\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 1\t\r \u00a0 2\t\r \u00a0 4\t\r \u00a0 0\t\r \u00a0 1\t\r \u00a0 2\t\r \u00a0 4\t\r \u00a0 5\t\r \u00a0 4\t\r \u00a0 9\t\r \u00a0 1\t\r \u00a0 1\t\r \u00a0 4\t\r \u00a0 2\t\r \u00a0 58\t\r \u00a0PKC\u03b7\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 1\t\r \u00a0 4\t\r \u00a0 0\t\r \u00a0 2\t\r \u00a0 4\t\r \u00a0 1\t\r \u00a0 5\t\r \u00a0 1\t\r \u00a0 2\t\r \u00a0 5\t\r \u00a0 6\t\r \u00a0 5\t\r \u00a0 5\t\r \u00a0 3\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 2\t\r \u00a0 2\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 56\t\r \u00a0PSKH1\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 1\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 1\t\r \u00a0 6\t\r \u00a0 5\t\r \u00a0 2\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 2\t\r \u00a0 4\t\r \u00a0 4\t\r \u00a0 2\t\r \u00a0 2\t\r \u00a0 2\t\r \u00a0 0\t\r \u00a0 3\t\r \u00a0 4\t\r \u00a0 8\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 1\t\r \u00a0 56\t\r \u00a0ROCK2\t\r \u00a0 8\t\r \u00a0 4\t\r \u00a0 6\t\r \u00a0 5\t\r \u00a0 5\t\r \u00a0 3\t\r \u00a0 3\t\r \u00a0 1\t\r \u00a0 2\t\r \u00a0 2\t\r \u00a0 0\t\r \u00a0 1\t\r \u00a0 3\t\r \u00a0 1\t\r \u00a0 3\t\r \u00a0 1\t\r \u00a0 3\t\r \u00a0 0\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 2\t\r \u00a0 0\t\r \u00a0 2\t\r \u00a0 0\t\r \u00a0 56\t\r \u00a0JNK2\t\r \u00a0 6\t\r \u00a0 5\t\r \u00a0 6\t\r \u00a0 7\t\r \u00a0 5\t\r \u00a0 0\t\r \u00a0 1\t\r \u00a0 5\t\r \u00a0 1\t\r \u00a0 1\t\r \u00a0 3\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 2\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 3\t\r \u00a0 0\t\r \u00a0 1\t\r \u00a0 1\t\r \u00a0 1\t\r \u00a0 3\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 1\t\r \u00a0 54\t\r \u00a0SGK\t\r \u00a0 1\t\r \u00a0 1\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 1\t\r \u00a0 1\t\r \u00a0 3\t\r \u00a0 4\t\r \u00a0 5\t\r \u00a0 4\t\r \u00a0 2\t\r \u00a0 6\t\r \u00a0 6\t\r \u00a0 4\t\r \u00a0 3\t\r \u00a0 5\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 2\t\r \u00a0 2\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 1\t\r \u00a0 54\t\r \u00a0PCTAIRE3\t\r \u00a0 9\t\r \u00a0 1\t\r \u00a0 4\t\r \u00a0 3\t\r \u00a0 2\t\r \u00a0 2\t\r \u00a0 3\t\r \u00a0 4\t\r \u00a0 1\t\r \u00a0 4\t\r \u00a0 1\t\r \u00a0 0\t\r \u00a0 2\t\r \u00a0 4\t\r \u00a0 2\t\r \u00a0 5\t\r \u00a0 0\t\r \u00a0 1\t\r \u00a0 2\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 0\t\r \u00a0 50\t\r \u00a0\t\r \u00a0 \t\r \u00a0\t\r \u00a0 127\t\r \u00a0 4.3.6\t\r \u00a0Phosphorylation\t\r \u00a0dynamics\t\r \u00a0following\t\r \u00a0WT\t\r \u00a0versus\t\r \u00a0\u2206sopB\t\r \u00a0infection\t\r \u00a0SopB\t\r \u00a0is\t\r \u00a0known\t\r \u00a0to\t\r \u00a0regulate\t\r \u00a0a\t\r \u00a0myriad\t\r \u00a0of\t\r \u00a0processes\t\r \u00a0within\t\r \u00a0host\t\r \u00a0cells.\t\r \u00a0These\t\r \u00a0include\t\r \u00a0formation\t\r \u00a0and\t\r \u00a0trafficking\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV\t\r \u00a0and\t\r \u00a0activation\t\r \u00a0of\t\r \u00a0Rho\t\r \u00a0GTPases\t\r \u00a0(Cdc42\t\r \u00a0and\t\r \u00a0RhoG)\t\r \u00a0and\t\r \u00a0Akt,\t\r \u00a0almost\t\r \u00a0exclusively\t\r \u00a0through\t\r \u00a0the\t\r \u00a0action\t\r \u00a0of\t\r \u00a0two\t\r \u00a0phosphoinositide\t\r \u00a0phosphatase\t\r \u00a0domains\t\r \u00a0located\t\r \u00a0near\t\r \u00a0its\t\r \u00a0C-\u00ad\u2010terminus(49,\t\r \u00a0163,\t\r \u00a0165,\t\r \u00a0185,\t\r \u00a0187,\t\r \u00a0197).\t\r \u00a0SopB\t\r \u00a0also\t\r \u00a0acts\t\r \u00a0in\t\r \u00a0a\t\r \u00a0redundant\t\r \u00a0manner\t\r \u00a0with\t\r \u00a0SopE\/E2\t\r \u00a0to\t\r \u00a0induce\t\r \u00a0all\t\r \u00a0of\t\r \u00a0the\t\r \u00a0innate\t\r \u00a0immune\t\r \u00a0signaling\t\r \u00a0detected\t\r \u00a0within\t\r \u00a0host\t\r \u00a0epithelial\t\r \u00a0cells(189).\t\r \u00a0Thus,\t\r \u00a0through\t\r \u00a0its\t\r \u00a0actions\t\r \u00a0on\t\r \u00a0Cdc42\t\r \u00a0and\t\r \u00a0Akt,\t\r \u00a0we\t\r \u00a0hypothesized\t\r \u00a0that\t\r \u00a0SopB\t\r \u00a0is\t\r \u00a0responsible\t\r \u00a0for\t\r \u00a0a\t\r \u00a0large\t\r \u00a0fraction\t\r \u00a0of\t\r \u00a0host\t\r \u00a0signaling\t\r \u00a0that\t\r \u00a0is\t\r \u00a0regulated\t\r \u00a0by\t\r \u00a0Salmonella.\t\r \u00a0Globally,\t\r \u00a0infection\t\r \u00a0with\t\r \u00a0the\t\r \u00a0\u0394sopB\t\r \u00a0strain\t\r \u00a0resulted\t\r \u00a0in\t\r \u00a0a\t\r \u00a035%\t\r \u00a0reduction\t\r \u00a0in\t\r \u00a0the\t\r \u00a0number\t\r \u00a0of\t\r \u00a0regulated\t\r \u00a0(P<.01)\t\r \u00a0phosphopeptides\t\r \u00a0relative\t\r \u00a0to\t\r \u00a0WT\t\r \u00a0(Figure\t\r \u00a04.5A),\t\r \u00a0and\t\r \u00a0this\t\r \u00a0was\t\r \u00a0consistent\t\r \u00a0for\t\r \u00a0pS\t\r \u00a0and\t\r \u00a0pT\t\r \u00a0sites\t\r \u00a0in\t\r \u00a0all\t\r \u00a0three\t\r \u00a0subcellular\t\r \u00a0fractions\t\r \u00a0tested.\t\r \u00a0Intriguingly,\t\r \u00a0\u2206sopB\t\r \u00a0infection\t\r \u00a0resulted\t\r \u00a0in\t\r \u00a0less\t\r \u00a0dynamic\t\r \u00a0pY\t\r \u00a0sites\t\r \u00a0detectable\t\r \u00a0in\t\r \u00a0the\t\r \u00a0membrane\t\r \u00a0fraction,\t\r \u00a0and\t\r \u00a0more\t\r \u00a0dynamic\t\r \u00a0pY\t\r \u00a0detectable\t\r \u00a0in\t\r \u00a0the\t\r \u00a0cytosolic\t\r \u00a0fraction.\t\r \u00a0SopB\t\r \u00a0is\t\r \u00a0known\t\r \u00a0to\t\r \u00a0localize\t\r \u00a0to\t\r \u00a0the\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0membrane\t\r \u00a0and\t\r \u00a0to\t\r \u00a0the\t\r \u00a0SCV,\t\r \u00a0and\t\r \u00a0likely\t\r \u00a0has\t\r \u00a0a\t\r \u00a0profound\t\r \u00a0local\t\r \u00a0effect\t\r \u00a0on\t\r \u00a0signaling\t\r \u00a0at\t\r \u00a0these\t\r \u00a0locations(197,\t\r \u00a0458).\t\r \u00a0SopB\t\r \u00a0can\t\r \u00a0also\t\r \u00a0recruit\t\r \u00a0proteins\t\r \u00a0to\t\r \u00a0these\t\r \u00a0compartments,\t\r \u00a0which\t\r \u00a0could\t\r \u00a0explain\t\r \u00a0the\t\r \u00a0reduced\t\r \u00a0regulation\t\r \u00a0observed\t\r \u00a0in\t\r \u00a0the\t\r \u00a0cytosol(58).\t\r \u00a0Initially,\t\r \u00a0SILAC\t\r \u00a0profiles\t\r \u00a0for\t\r \u00a0all\t\r \u00a0known\t\r \u00a0host\t\r \u00a0phosphorylation\t\r \u00a0targets\t\r \u00a0were\t\r \u00a0compared\t\r \u00a0during\t\r \u00a0infection\t\r \u00a0with\t\r \u00a0the\t\r \u00a0WT\t\r \u00a0and\t\r \u00a0\u0394sopB\t\r \u00a0strains.\t\r \u00a0As\t\r \u00a0expected,\t\r \u00a0due\t\r \u00a0to\t\r \u00a0redundancy\t\r \u00a0in\t\r \u00a0SopB\t\r \u00a0and\t\r \u00a0SopE\/E2\t\r \u00a0function,\t\r \u00a0phosphorylation\t\r \u00a0dynamics\t\r \u00a0for\t\r \u00a0ERK2,\t\r \u00a0ATF2\t\r \u00a0and\t\r \u00a07,\t\r \u00a0and\t\r \u00a0PAK4\t\r \u00a0were\t\r \u00a0reduced,\t\r \u00a0but\t\r \u00a0not\t\r \u00a0eliminated,\t\r \u00a0following\t\r \u00a0infection\t\r \u00a0with\t\r \u00a0the\t\r \u00a0\u0394sopB\t\r \u00a0strain.\t\r \u00a0Activating\t\r \u00a0sites\t\r \u00a0in\t\r \u00a0both\t\r \u00a0Ribosomal\t\r \u00a0protein\t\r \u00a0S6\t\r \u00a0kinase\t\r \u00a0alpha-\u00ad\u20101\t\r \u00a0 \t\r \u00a0\t\r \u00a0 128\t\r \u00a0 (RSK1)\t\r \u00a0and\t\r \u00a0alpha-\u00ad\u20104\t\r \u00a0(MSK2)\t\r \u00a0were\t\r \u00a0also\t\r \u00a0up-\u00ad\u2010regulated\t\r \u00a0four-\u00ad\u2010\t\r \u00a0and\t\r \u00a0ten-\u00ad\u2010fold\t\r \u00a0during\t\r \u00a0infection\t\r \u00a0with\t\r \u00a0WT\t\r \u00a0but\t\r \u00a0not\t\r \u00a0the\t\r \u00a0\u0394sopB\t\r \u00a0strain\t\r \u00a0(Figure\t\r \u00a04.5B).\t\r \u00a0Ser-\u00ad\u2010368\t\r \u00a0from\t\r \u00a0RSK1\t\r \u00a0is\t\r \u00a0an\t\r \u00a0activating\t\r \u00a0site\t\r \u00a0phosphorylated\t\r \u00a0by\t\r \u00a0ERK1\/2,\t\r \u00a0while\t\r \u00a0Ser-\u00ad\u2010343\t\r \u00a0and\t\r \u00a0Ser-\u00ad\u2010347\t\r \u00a0from\t\r \u00a0MSK2\t\r \u00a0are\t\r \u00a0phosphorylated\t\r \u00a0by\t\r \u00a0p38\t\r \u00a0MAPK(525,\t\r \u00a0530).\t\r \u00a0In\t\r \u00a0contrast\t\r \u00a0to\t\r \u00a0the\t\r \u00a0proteins\t\r \u00a0mentioned\t\r \u00a0above,\t\r \u00a0it\t\r \u00a0appears\t\r \u00a0that\t\r \u00a0activation\t\r \u00a0of\t\r \u00a0RSK1\t\r \u00a0and\t\r \u00a0MSK2\t\r \u00a0is\t\r \u00a0completely\t\r \u00a0dependent\t\r \u00a0on\t\r \u00a0SopB.\t\r \u00a0This\t\r \u00a0indicates\t\r \u00a0that\t\r \u00a0SopB\t\r \u00a0and\t\r \u00a0SopE\/E2\t\r \u00a0may\t\r \u00a0in\t\r \u00a0fact\t\r \u00a0target\t\r \u00a0MAPKs\t\r \u00a0differently,\t\r \u00a0perhaps\t\r \u00a0through\t\r \u00a0signaling\t\r \u00a0downstream\t\r \u00a0of\t\r \u00a0Akt.\t\r \u00a0Four\t\r \u00a0MAPK\t\r \u00a0cascades\t\r \u00a0have\t\r \u00a0been\t\r \u00a0well-\u00ad\u2010described:\t\r \u00a0the\t\r \u00a0ERK1\/2\t\r \u00a0pathway,\t\r \u00a0the\t\r \u00a0JNK\t\r \u00a0pathway,\t\r \u00a0the\t\r \u00a0p38\t\r \u00a0pathway,\t\r \u00a0and\t\r \u00a0the\t\r \u00a0ERK5\t\r \u00a0pathway(533).\t\r \u00a0All\t\r \u00a0are\t\r \u00a0thought\t\r \u00a0to\t\r \u00a0regulate\t\r \u00a0cellular\t\r \u00a0growth\t\r \u00a0and\t\r \u00a0proliferation,\t\r \u00a0but\t\r \u00a0much\t\r \u00a0less\t\r \u00a0is\t\r \u00a0known\t\r \u00a0about\t\r \u00a0the\t\r \u00a0signaling\t\r \u00a0pathways\t\r \u00a0leading\t\r \u00a0to\t\r \u00a0ERK5\t\r \u00a0activation\t\r \u00a0compared\t\r \u00a0to\t\r \u00a0the\t\r \u00a0other\t\r \u00a0three,\t\r \u00a0and\t\r \u00a0only\t\r \u00a0ERK1\/2,\t\r \u00a0JNK\t\r \u00a0and\t\r \u00a0p38\t\r \u00a0have\t\r \u00a0been\t\r \u00a0implicated\t\r \u00a0in\t\r \u00a0Salmonella\t\r \u00a0infection(534).\t\r \u00a0However,\t\r \u00a0two\t\r \u00a0activating\t\r \u00a0autophosphorylation\t\r \u00a0sites\t\r \u00a0(Ser731\t\r \u00a0and\t\r \u00a0Thr733)\t\r \u00a0from\t\r \u00a0ERK5\t\r \u00a0were\t\r \u00a0up-\u00ad\u2010regulated\t\r \u00a0more\t\r \u00a0than\t\r \u00a0four-\u00ad\u2010fold\t\r \u00a0during\t\r \u00a0infection\t\r \u00a0with\t\r \u00a0the\t\r \u00a0\u0394sopB\t\r \u00a0strain\t\r \u00a0(Figure\t\r \u00a04.5B)(535).\t\r \u00a0While\t\r \u00a0this\t\r \u00a0shows\t\r \u00a0distinct\t\r \u00a0regulation\t\r \u00a0of\t\r \u00a0ERK5\t\r \u00a0by\t\r \u00a0Salmonella,\t\r \u00a0ERK5\t\r \u00a0was\t\r \u00a0not\t\r \u00a0identified\t\r \u00a0in\t\r \u00a0the\t\r \u00a0WT\t\r \u00a0dataset,\t\r \u00a0so\t\r \u00a0it\t\r \u00a0is\t\r \u00a0currently\t\r \u00a0unclear\t\r \u00a0whether\t\r \u00a0ERK5\t\r \u00a0is\t\r \u00a0up-\u00ad\u2010regulated\t\r \u00a0by\t\r \u00a0WT,\t\r \u00a0or\t\r \u00a0potentially\t\r \u00a0down-\u00ad\u2010regulated\t\r \u00a0by\t\r \u00a0SopB.\t\r \u00a0Presumably\t\r \u00a0not\t\r \u00a0all\t\r \u00a0the\t\r \u00a0proteins\t\r \u00a0whose\t\r \u00a0regulation\t\r \u00a0is\t\r \u00a0suppressed\t\r \u00a0with\t\r \u00a0\u2206sopB\t\r \u00a0are\t\r \u00a0actually\t\r \u00a0direct\t\r \u00a0targets\t\r \u00a0of\t\r \u00a0SopB;\t\r \u00a0rather,\t\r \u00a0it\t\r \u00a0is\t\r \u00a0more\t\r \u00a0likely\t\r \u00a0that\t\r \u00a0SopB\t\r \u00a0acts\t\r \u00a0through\t\r \u00a0a\t\r \u00a0small\t\r \u00a0number\t\r \u00a0other\t\r \u00a0kinases\t\r \u00a0or\t\r \u00a0phosphatases\t\r \u00a0to\t\r \u00a0impact\t\r \u00a0a\t\r \u00a0wider\t\r \u00a0range\t\r \u00a0of\t\r \u00a0indirect\t\r \u00a0targets.\t\r \u00a0To\t\r \u00a0identify\t\r \u00a0kinases\t\r \u00a0that\t\r \u00a0might\t\r \u00a0be\t\r \u00a0targeted\t\r \u00a0by\t\r \u00a0SopB,\t\r \u00a0we\t\r \u00a0first\t\r \u00a0filtered\t\r \u00a0out\t\r \u00a0all\t\r \u00a0phosphopeptides\t\r \u00a0that\t\r \u00a0showed\t\r \u00a0at\t\r \u00a0least\t\r \u00a0a\t\r \u00a0two-\u00ad\u2010fold\t\r \u00a0difference\t\r \u00a0between\t\r \u00a0WT\t\r \u00a0and\t\r \u00a0\u2206sopB\t\r \u00a0infections,\t\r \u00a0and\t\r \u00a0that\t\r \u00a0were\t\r \u00a0significantly\t\r \u00a0different\t\r \u00a0(P<.01)\t\r \u00a0from\t\r \u00a0mock\t\r \u00a0infections.\t\r \u00a0 \t\r \u00a0\t\r \u00a0 129\t\r \u00a0 Among\t\r \u00a0the\t\r \u00a0348\t\r \u00a0phosphopeptides\t\r \u00a0meeting\t\r \u00a0this\t\r \u00a0criteria\t\r \u00a0one\t\r \u00a0sequence\t\r \u00a0motif,\t\r \u00a0RxRxxS,\t\r \u00a0was\t\r \u00a0significantly\t\r \u00a0enriched,\t\r \u00a0occurring\t\r \u00a0in\t\r \u00a015\t\r \u00a0peptides\t\r \u00a0(Figure\t\r \u00a04.5C).\t\r \u00a0This\t\r \u00a0motif\t\r \u00a0is\t\r \u00a0a\t\r \u00a0consensus\t\r \u00a0recognition\t\r \u00a0phosphorylation\t\r \u00a0site\t\r \u00a0for\t\r \u00a0Akt,\t\r \u00a0RSK\t\r \u00a0and\t\r \u00a0Ribosomal\t\r \u00a0protein\t\r \u00a0S6\t\r \u00a0kinase\t\r \u00a0beta\t\r \u00a0(p70S6K)(529),\t\r \u00a0and\t\r \u00a0at\t\r \u00a0least\t\r \u00a07\t\r \u00a0of\t\r \u00a0the\t\r \u00a015\t\r \u00a0peptides\t\r \u00a0are\t\r \u00a0known\t\r \u00a0substrates\t\r \u00a0of\t\r \u00a0one\t\r \u00a0or\t\r \u00a0more\t\r \u00a0of\t\r \u00a0these\t\r \u00a0kinases\t\r \u00a0(Figure\t\r \u00a04.5C)(529).\t\r \u00a0These\t\r \u00a0data\t\r \u00a0indicate\t\r \u00a0that\t\r \u00a0SopB's\t\r \u00a0effects\t\r \u00a0are\t\r \u00a0centered\t\r \u00a0around\t\r \u00a0Akt\t\r \u00a0and\t\r \u00a0also\t\r \u00a0RSK1\t\r \u00a0(Figure\t\r \u00a04.5B),\t\r \u00a0which\t\r \u00a0in\t\r \u00a0turn\t\r \u00a0targets\t\r \u00a0Sodium\/hydrogen\t\r \u00a0exchanger\t\r \u00a01\t\r \u00a0(NHE1)\t\r \u00a0(Figure\t\r \u00a05C)(66,\t\r \u00a0536).\t\r \u00a0Beyond\t\r \u00a0this,\t\r \u00a0however,\t\r \u00a0deeper\t\r \u00a0probing\t\r \u00a0into\t\r \u00a0specific\t\r \u00a0phosphorylation\t\r \u00a0events\t\r \u00a0can\t\r \u00a0provide\t\r \u00a0mechanistic\t\r \u00a0insights\t\r \u00a0into\t\r \u00a0previously\t\r \u00a0unexplained\t\r \u00a0observations\t\r \u00a0associated\t\r \u00a0with\t\r \u00a0Salmonella\t\r \u00a0infection.\t\r \u00a0SopB\t\r \u00a0and\t\r \u00a0Apoptosis\t\r \u00a0SopB\t\r \u00a0is\t\r \u00a0known\t\r \u00a0to\t\r \u00a0down-\u00ad\u2010regulate\t\r \u00a0apoptosis\t\r \u00a0through\t\r \u00a0an\t\r \u00a0unknown,\t\r \u00a0Akt-\u00ad\u2010dependent\t\r \u00a0mechanism.\t\r \u00a0Consistent\t\r \u00a0with\t\r \u00a0this,\t\r \u00a0we\t\r \u00a0detect\t\r \u00a0a\t\r \u00a02.5-\u00ad\u2010fold\t\r \u00a0increase\t\r \u00a0in\t\r \u00a0phosphorylation\t\r \u00a0of\t\r \u00a0Ser99\t\r \u00a0of\t\r \u00a0Bcl2\t\r \u00a0antagonist\t\r \u00a0of\t\r \u00a0cell\t\r \u00a0death\t\r \u00a0(BAD)\t\r \u00a0with\t\r \u00a0infection\t\r \u00a0by\t\r \u00a0WT\t\r \u00a0bacteria\t\r \u00a0and\t\r \u00a0this\t\r \u00a0increase\t\r \u00a0is\t\r \u00a0completely\t\r \u00a0abrogated\t\r \u00a0with\t\r \u00a0\u2206sopB\t\r \u00a0(Figure\t\r \u00a04.5D).\t\r \u00a0Ser99\t\r \u00a0of\t\r \u00a0BAD\t\r \u00a0is\t\r \u00a0phosphorylated\t\r \u00a0by\t\r \u00a0Akt,\t\r \u00a0resulting\t\r \u00a0in\t\r \u00a0BAD\t\r \u00a0being\t\r \u00a0sequestered\t\r \u00a0in\t\r \u00a0the\t\r \u00a0cytosol\t\r \u00a0and\t\r \u00a0thereby\t\r \u00a0inactivating\t\r \u00a0its\t\r \u00a0pro-\u00ad\u2010apoptotic\t\r \u00a0activity\t\r \u00a0by\t\r \u00a0preventing\t\r \u00a0its\t\r \u00a0association\t\r \u00a0with\t\r \u00a0pro-\u00ad\u2010survival\t\r \u00a0proteins\t\r \u00a0at\t\r \u00a0mitochondria\t\r \u00a0(521).\t\r \u00a0Thus,\t\r \u00a0SopB\t\r \u00a0and\t\r \u00a0Akt\t\r \u00a0dependent\t\r \u00a0anti-\u00ad\u2010apoptotic\t\r \u00a0signaling\t\r \u00a0is\t\r \u00a0likely\t\r \u00a0facilitated\t\r \u00a0in\t\r \u00a0part\t\r \u00a0through\t\r \u00a0BAD\t\r \u00a0inactivation.\t\r \u00a0SopB\t\r \u00a0and\t\r \u00a0actin\t\r \u00a0dynamics\t\r \u00a0 Salmonella\t\r \u00a0effector\t\r \u00a0proteins\t\r \u00a0induce\t\r \u00a0a\t\r \u00a0massive\t\r \u00a0rearrangement\t\r \u00a0of\t\r \u00a0actin\t\r \u00a0during\t\r \u00a0the\t\r \u00a0invasion\t\r \u00a0process,\t\r \u00a0which\t\r \u00a0is\t\r \u00a0generally\t\r \u00a0known\t\r \u00a0to\t\r \u00a0work\t\r \u00a0through\t\r \u00a0the\t\r \u00a0activity\t\r \u00a0of\t\r \u00a0SopB\t\r \u00a0 \t\r \u00a0\t\r \u00a0 130\t\r \u00a0 and\t\r \u00a0SopE\/E2\t\r \u00a0on\t\r \u00a0small\t\r \u00a0G\t\r \u00a0proteins.\t\r \u00a0From\t\r \u00a0other\t\r \u00a0work,\t\r \u00a0Akt\t\r \u00a0phosphorylation\t\r \u00a0of\t\r \u00a0Rac1\t\r \u00a0at\t\r \u00a0Ser-\u00ad\u201071\t\r \u00a0inhibits\t\r \u00a0GTP\t\r \u00a0binding\t\r \u00a0and\t\r \u00a0thus\t\r \u00a0Rac1\t\r \u00a0activity(527);\t\r \u00a0when\t\r \u00a0active,\t\r \u00a0however,\t\r \u00a0Rac1\t\r \u00a0can\t\r \u00a0signal\t\r \u00a0through\t\r \u00a0PAK\t\r \u00a0and\t\r \u00a0LIM\t\r \u00a0domain\t\r \u00a0kinase\t\r \u00a01\t\r \u00a0(LIMK)\t\r \u00a0to\t\r \u00a0phosphorylate\t\r \u00a0Ser-\u00ad\u20103\t\r \u00a0of\t\r \u00a0cofilin\t\r \u00a0and\t\r \u00a0thereby\t\r \u00a0inhibit\t\r \u00a0its\t\r \u00a0actin\t\r \u00a0depolymerizing\t\r \u00a0activity(522,\t\r \u00a0523).\t\r \u00a0Infection\t\r \u00a0with\t\r \u00a0WT\t\r \u00a0Salmonella\t\r \u00a0induces\t\r \u00a0a\t\r \u00a03.5-\u00ad\u2010fold\t\r \u00a0increase\t\r \u00a0in\t\r \u00a0Ser-\u00ad\u201071\t\r \u00a0phosphorylation\t\r \u00a0of\t\r \u00a0Rac1\t\r \u00a0and\t\r \u00a0a\t\r \u00a02-\u00ad\u2010fold\t\r \u00a0decrease\t\r \u00a0in\t\r \u00a0Ser-\u00ad\u20103\t\r \u00a0phosphorylation\t\r \u00a0of\t\r \u00a0cofilin\t\r \u00a0(Figure\t\r \u00a04.5D,\t\r \u00a0confirmed\t\r \u00a0by\t\r \u00a0immunoblotting\t\r \u00a0in\t\r \u00a0Figure\t\r \u00a04.3A).\t\r \u00a0Both\t\r \u00a0of\t\r \u00a0these\t\r \u00a0effects\t\r \u00a0were\t\r \u00a0completely\t\r \u00a0dependent\t\r \u00a0on\t\r \u00a0SopB,\t\r \u00a0indicating\t\r \u00a0that,\t\r \u00a0through\t\r \u00a0Akt,\t\r \u00a0SopB\t\r \u00a0can\t\r \u00a0inactivate\t\r \u00a0Rac1\t\r \u00a0to\t\r \u00a0promote\t\r \u00a0cofilin\t\r \u00a0activity.\t\r \u00a0SopB\t\r \u00a0and\t\r \u00a0SCV\t\r \u00a0trafficking\t\r \u00a0Recruitment\t\r \u00a0of\t\r \u00a0VAMP8\t\r \u00a0positive\t\r \u00a0endocytic\t\r \u00a0vesicles\t\r \u00a0to\t\r \u00a0Salmonella-\u00ad\u2010induced\t\r \u00a0membrane\t\r \u00a0ruffles\t\r \u00a0is\t\r \u00a0required\t\r \u00a0for\t\r \u00a0efficient\t\r \u00a0invasion,\t\r \u00a0and\t\r \u00a0dependent\t\r \u00a0on\t\r \u00a0the\t\r \u00a0phosphatase\t\r \u00a0activity\t\r \u00a0of\t\r \u00a0SopB(50).\t\r \u00a0However,\t\r \u00a0while\t\r \u00a0these\t\r \u00a0vesicles\t\r \u00a0likely\t\r \u00a0provide\t\r \u00a0membranes\t\r \u00a0to\t\r \u00a0the\t\r \u00a0forming\t\r \u00a0macropinosome,\t\r \u00a0targets\t\r \u00a0of\t\r \u00a0VAMP8\t\r \u00a0during\t\r \u00a0infection\t\r \u00a0are\t\r \u00a0currently\t\r \u00a0unknown.\t\r \u00a0Syntaxin\t\r \u00a07\t\r \u00a0(STX7)\t\r \u00a0forms\t\r \u00a0a\t\r \u00a0SNARE\t\r \u00a0complex\t\r \u00a0with\t\r \u00a0VTI1b,\t\r \u00a0syntaxin\t\r \u00a08\t\r \u00a0and\t\r \u00a0VAMP8\t\r \u00a0to\t\r \u00a0fuse\t\r \u00a0membranes\t\r \u00a0within\t\r \u00a0the\t\r \u00a0endosomal\t\r \u00a0pathway(537):\t\r \u00a0formation\t\r \u00a0of\t\r \u00a0this\t\r \u00a0complex\t\r \u00a0can\t\r \u00a0be\t\r \u00a0positively\t\r \u00a0regulated\t\r \u00a0by\t\r \u00a0Akt,\t\r \u00a0through\t\r \u00a0phosphorylation\t\r \u00a0of\t\r \u00a0syntaxin\t\r \u00a07\t\r \u00a0on\t\r \u00a0Ser-\u00ad\u2010126\t\r \u00a0and\t\r \u00a0Ser-\u00ad\u2010129(538).\t\r \u00a0Intriguingly,\t\r \u00a0Salmonella\t\r \u00a0induces\t\r \u00a0a\t\r \u00a0SopB-\u00ad\u2010dependent\t\r \u00a0three-\u00ad\u2010fold\t\r \u00a0up-\u00ad\u2010regulation\t\r \u00a0of\t\r \u00a0phosphorylation\t\r \u00a0on\t\r \u00a0both\t\r \u00a0Ser-\u00ad\u2010126\t\r \u00a0and\t\r \u00a0Ser-\u00ad\u2010129\t\r \u00a0(Figure\t\r \u00a04.5D).\t\r \u00a0Thus,\t\r \u00a0SopB\t\r \u00a0likely\t\r \u00a0promotes\t\r \u00a0SNARE\t\r \u00a0complex\t\r \u00a0formation\t\r \u00a0through\t\r \u00a0VAMP8\t\r \u00a0recruitment,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0Akt\t\r \u00a0dependent\t\r \u00a0phosphorylation\t\r \u00a0of\t\r \u00a0STX7\t\r \u00a0to\t\r \u00a0regulate\t\r \u00a0vesicle\t\r \u00a0fusion\t\r \u00a0during\t\r \u00a0formation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV.\t\r \u00a0 \t\r \u00a0\t\r \u00a0 131\t\r \u00a0 Figure\t\r \u00a04.5\t\r \u00a0Novel\t\r \u00a0host\t\r \u00a0targets\t\r \u00a0of\t\r \u00a0the\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0effector\t\r \u00a0SopB\t\r \u00a0 \t\r \u00a0A)\t\r \u00a0Global\t\r \u00a0impact\t\r \u00a0of\t\r \u00a0SopB\t\r \u00a0on\t\r \u00a0host\t\r \u00a0phosphorylation.\t\r \u00a0The\t\r \u00a0percentage\t\r \u00a0of\t\r \u00a0pS,\t\r \u00a0pT\t\r \u00a0and\t\r \u00a0pY\t\r \u00a0sites\t\r \u00a0regulated\t\r \u00a0during\t\r \u00a0WT\t\r \u00a0and\t\r \u00a0\u2206sopB\t\r \u00a0infection\t\r \u00a0are\t\r \u00a0shown\t\r \u00a0for\t\r \u00a0each\t\r \u00a0subcellular\t\r \u00a0fraction.\t\r \u00a0B)\t\r \u00a0Novel\t\r \u00a0MAPK\t\r \u00a0targets\t\r \u00a0of\t\r \u00a0SopB.\t\r \u00a0SILAC\t\r \u00a0profiles\t\r \u00a0are\t\r \u00a0shown\t\r \u00a0for\t\r \u00a0the\t\r \u00a0indicated\t\r \u00a0phosphopeptides\t\r \u00a0from\t\r \u00a0RSK1,\t\r \u00a0MSK2\t\r \u00a0and\t\r \u00a0ERK5\t\r \u00a0during\t\r \u00a0infection\t\r \u00a0with\t\r \u00a0WT\t\r \u00a0and\t\r \u00a0\u2206sopB.\t\r \u00a0C)\t\r \u00a0Amino\t\r \u00a0acid\t\r \u00a0sequences\t\r \u00a0surrounding\t\r \u00a0SopB\t\r \u00a0regulated\t\r \u00a0 \t\r \u00a0\t\r \u00a0 132\t\r \u00a0 phosphosites\t\r \u00a0that\t\r \u00a0feature\t\r \u00a0a\t\r \u00a0RxRxxS\t\r \u00a0motif.\t\r \u00a0The\t\r \u00a0gene\t\r \u00a0names,\t\r \u00a0Unitprot\t\r \u00a0identification\t\r \u00a0numbers\t\r \u00a0and\t\r \u00a0location\t\r \u00a0of\t\r \u00a0the\t\r \u00a0phosphosite\t\r \u00a0are\t\r \u00a0shown,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0known\t\r \u00a0kinases\t\r \u00a0targeting\t\r \u00a0each\t\r \u00a0site.\t\r \u00a0D)\t\r \u00a0Novel\t\r \u00a0host\t\r \u00a0targets\t\r \u00a0of\t\r \u00a0SopB.\t\r \u00a0SILAC\t\r \u00a0profiles\t\r \u00a0are\t\r \u00a0shown\t\r \u00a0for\t\r \u00a0the\t\r \u00a0indicated\t\r \u00a0phosphopeptides\t\r \u00a0from\t\r \u00a0SopB\t\r \u00a0regulated\t\r \u00a0proteins\t\r \u00a0containing\t\r \u00a0the\t\r \u00a0RxRxxS\t\r \u00a0motif.\t\r \u00a0In\t\r \u00a0both\t\r \u00a0A\t\r \u00a0and\t\r \u00a0D,\t\r \u00a0phosphorylated\t\r \u00a0residues\t\r \u00a0are\t\r \u00a0shown\t\r \u00a0in\t\r \u00a0red.\t\r \u00a0\t\r \u00a0\t\r \u00a0 4.4\t\r \u00a0Discussion\t\r \u00a0The\t\r \u00a0mechanism(s)\t\r \u00a0through\t\r \u00a0which\t\r \u00a0Salmonella\t\r \u00a0effector\t\r \u00a0proteins\t\r \u00a0bypass\t\r \u00a0innate\t\r \u00a0immune\t\r \u00a0receptors\t\r \u00a0to\t\r \u00a0exploit\t\r \u00a0host\t\r \u00a0signaling\t\r \u00a0pathways\t\r \u00a0represents\t\r \u00a0a\t\r \u00a0unique\t\r \u00a0mode\t\r \u00a0of\t\r \u00a0cell\t\r \u00a0signaling\t\r \u00a0and\t\r \u00a0an\t\r \u00a0exceptional\t\r \u00a0level\t\r \u00a0of\t\r \u00a0adaptation\t\r \u00a0by\t\r \u00a0Salmonella\t\r \u00a0to\t\r \u00a0its\t\r \u00a0host.\t\r \u00a0Here\t\r \u00a0we\t\r \u00a0present\t\r \u00a0a\t\r \u00a0global\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0host\t\r \u00a0phosphorylation\t\r \u00a0events\t\r \u00a0induced\t\r \u00a0during\t\r \u00a0the\t\r \u00a0initial\t\r \u00a0phase\t\r \u00a0of\t\r \u00a0S.\t\r \u00a0Typhimurium\t\r \u00a0challenge\t\r \u00a0of\t\r \u00a0a\t\r \u00a0well\t\r \u00a0characterized\t\r \u00a0human\t\r \u00a0epithelial\t\r \u00a0cell\t\r \u00a0line,\t\r \u00a0allowing\t\r \u00a0temporal\t\r \u00a0modeling\t\r \u00a0of\t\r \u00a0how\t\r \u00a0more\t\r \u00a0than\t\r \u00a06800\t\r \u00a0phosphopeptides\t\r \u00a0respond\t\r \u00a0to\t\r \u00a0infection.\t\r \u00a0Given\t\r \u00a0that\t\r \u00a0several\t\r \u00a0Salmonella\t\r \u00a0effectors\t\r \u00a0activate\t\r \u00a0signaling\t\r \u00a0proteins\t\r \u00a0directly\t\r \u00a0and\t\r \u00a0at\t\r \u00a0various\t\r \u00a0stages\t\r \u00a0downstream\t\r \u00a0of\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0receptors,\t\r \u00a0the\t\r \u00a0data\t\r \u00a0here\t\r \u00a0represent\t\r \u00a0a\t\r \u00a0unique\t\r \u00a0system,\t\r \u00a0perhaps\t\r \u00a0not\t\r \u00a0surprisingly,\t\r \u00a0with\t\r \u00a0several\t\r \u00a0distinctions\t\r \u00a0to\t\r \u00a0classical\t\r \u00a0receptor-\u00ad\u2010induced\t\r \u00a0signaling\t\r \u00a0dynamics.\t\r \u00a0Despite\t\r \u00a0no\t\r \u00a0apparent\t\r \u00a0connection,\t\r \u00a0several\t\r \u00a0comparisons\t\r \u00a0have\t\r \u00a0been\t\r \u00a0made\t\r \u00a0between\t\r \u00a0signaling\t\r \u00a0induced\t\r \u00a0in\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0by\t\r \u00a0Salmonella\t\r \u00a0and\t\r \u00a0EGF.\t\r \u00a0Initially\t\r \u00a0it\t\r \u00a0was\t\r \u00a0proposed\t\r \u00a0that\t\r \u00a0Salmonella\t\r \u00a0use\t\r \u00a0the\t\r \u00a0EGF\t\r \u00a0receptor\t\r \u00a0(EGFR)\t\r \u00a0during\t\r \u00a0invasion,\t\r \u00a0but\t\r \u00a0neither\t\r \u00a0the\t\r \u00a0data\t\r \u00a0presented\t\r \u00a0here\t\r \u00a0nor\t\r \u00a0that\t\r \u00a0in\t\r \u00a0a\t\r \u00a0previous\t\r \u00a0study\t\r \u00a0could\t\r \u00a0detect\t\r \u00a0any\t\r \u00a0changes\t\r \u00a0in\t\r \u00a0EGFR\t\r \u00a0tyrosine\t\r \u00a0phosphorylation\t\r \u00a0after\t\r \u00a0Salmonella\t\r \u00a0invasion(32,\t\r \u00a0541).\t\r \u00a0At\t\r \u00a0a\t\r \u00a0gross\t\r \u00a0level,\t\r \u00a0EGF\t\r \u00a0and\t\r \u00a0Salmonella\t\r \u00a0have\t\r \u00a0some\t\r \u00a0similar\t\r \u00a0effects\t\r \u00a0and\t\r \u00a0modes\t\r \u00a0of\t\r \u00a0action:\t\r \u00a0both\t\r \u00a0activate\t\r \u00a0canonical\t\r \u00a0MAPK\t\r \u00a0signaling\t\r \u00a0pathways\t\r \u00a0within\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0and\t\r \u00a0induce\t\r \u00a0internalization\t\r \u00a0into\t\r \u00a0an\t\r \u00a0early\t\r \u00a0endosome\t\r \u00a0or\t\r \u00a0similar\t\r \u00a0compartment.\t\r \u00a0However,\t\r \u00a0our\t\r \u00a0data\t\r \u00a0demonstrate\t\r \u00a0clearly\t\r \u00a0that,\t\r \u00a0when\t\r \u00a0compared\t\r \u00a0to\t\r \u00a0EGF-\u00ad\u2010stimulated\t\r \u00a0signaling,\t\r \u00a0Salmonella-\u00ad\u2010induced\t\r \u00a0pY\t\r \u00a0signaling\t\r \u00a0 \t\r \u00a0\t\r \u00a0 133\t\r \u00a0 is\t\r \u00a0much\t\r \u00a0slower,\t\r \u00a0increasing\t\r \u00a0within\t\r \u00a0the\t\r \u00a0same\t\r \u00a0time\t\r \u00a0frame\t\r \u00a0as\t\r \u00a0pS\t\r \u00a0and\t\r \u00a0pT(297).\t\r \u00a0We\t\r \u00a0expect\t\r \u00a0that\t\r \u00a0this\t\r \u00a0is\t\r \u00a0a\t\r \u00a0result\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0signaling\t\r \u00a0being\t\r \u00a0initiated\t\r \u00a0at\t\r \u00a0intracellular\t\r \u00a0sites\t\r \u00a0where\t\r \u00a0effectors\t\r \u00a0act,\t\r \u00a0rather\t\r \u00a0than\t\r \u00a0at\t\r \u00a0the\t\r \u00a0membrane\t\r \u00a0as\t\r \u00a0in\t\r \u00a0the\t\r \u00a0case\t\r \u00a0of\t\r \u00a0receptor\t\r \u00a0tyrosine\t\r \u00a0kinase\t\r \u00a0pathways.\t\r \u00a0Indeed,\t\r \u00a0if\t\r \u00a0one\t\r \u00a0compares\t\r \u00a0individual\t\r \u00a0peptides\t\r \u00a0there\t\r \u00a0is\t\r \u00a0very\t\r \u00a0little\t\r \u00a0similarity\t\r \u00a0between\t\r \u00a0the\t\r \u00a0two\t\r \u00a0signaling\t\r \u00a0systems,\t\r \u00a0and,\t\r \u00a0where\t\r \u00a0similar\t\r \u00a0trends\t\r \u00a0are\t\r \u00a0seen,\t\r \u00a0they\t\r \u00a0are\t\r \u00a0almost\t\r \u00a0always\t\r \u00a0delayed\t\r \u00a0in\t\r \u00a0Salmonella\t\r \u00a0versus\t\r \u00a0EGF.\t\r \u00a0\t\r \u00a0Epithelial\t\r \u00a0cells\t\r \u00a0typically\t\r \u00a0down-\u00ad\u2010regulate\t\r \u00a0innate\t\r \u00a0immune\t\r \u00a0signaling\t\r \u00a0to\t\r \u00a0avoid\t\r \u00a0uncontrolled\t\r \u00a0inflammation,\t\r \u00a0but\t\r \u00a0Salmonella\t\r \u00a0effectors\t\r \u00a0SopB\t\r \u00a0and\t\r \u00a0SopE\/E2\t\r \u00a0initiate\t\r \u00a0innate\t\r \u00a0signaling\t\r \u00a0pathways\t\r \u00a0themselves,\t\r \u00a0independent\t\r \u00a0of\t\r \u00a0TLRs\t\r \u00a0and\t\r \u00a0NLRs(189).\t\r \u00a0Thus,\t\r \u00a0 Salmonella-\u00ad\u2010induced\t\r \u00a0signaling\t\r \u00a0events\t\r \u00a0in\t\r \u00a0HeLa\t\r \u00a0cells\t\r \u00a0are\t\r \u00a0more\t\r \u00a0likely\t\r \u00a0to\t\r \u00a0be\t\r \u00a0results\t\r \u00a0of\t\r \u00a0the\t\r \u00a0bacteria\u2019s\t\r \u00a0own\t\r \u00a0actions\t\r \u00a0rather\t\r \u00a0than\t\r \u00a0undesirable\t\r \u00a0side\t\r \u00a0effects\t\r \u00a0of\t\r \u00a0its\t\r \u00a0invasion,\t\r \u00a0and\t\r \u00a0the\t\r \u00a0regulated\t\r \u00a0phosphopeptides\t\r \u00a0detected\t\r \u00a0here\t\r \u00a0represent\t\r \u00a0potential\t\r \u00a0novel\t\r \u00a0host\t\r \u00a0targets\t\r \u00a0of\t\r \u00a0 Salmonella\t\r \u00a0(Table\t\r \u00a04.1).\t\r \u00a0Alongside\t\r \u00a0our\t\r \u00a0initial\t\r \u00a0large-\u00ad\u2010scale\t\r \u00a0phosphoproteomic\t\r \u00a0studies\t\r \u00a0using\t\r \u00a0WT\t\r \u00a0bacteria,\t\r \u00a0we\t\r \u00a0were\t\r \u00a0able\t\r \u00a0to\t\r \u00a0validate\t\r \u00a0more\t\r \u00a0than\t\r \u00a0a\t\r \u00a0third\t\r \u00a0of\t\r \u00a0these\t\r \u00a0using\t\r \u00a0a\t\r \u00a0\u2206sopB\t\r \u00a0strain.\t\r \u00a0In\t\r \u00a0addition\t\r \u00a0to\t\r \u00a0demonstrating\t\r \u00a0the\t\r \u00a0validity\t\r \u00a0of\t\r \u00a0the\t\r \u00a0initial\t\r \u00a0dataset,\t\r \u00a0the\t\r \u00a0results\t\r \u00a0with\t\r \u00a0the\t\r \u00a0mutant\t\r \u00a0strain\t\r \u00a0indicate\t\r \u00a0that\t\r \u00a0the\t\r \u00a0role\t\r \u00a0of\t\r \u00a0SopB\t\r \u00a0is\t\r \u00a0centered\t\r \u00a0around\t\r \u00a0Akt\t\r \u00a0and\t\r \u00a0RSK1\t\r \u00a0kinases,\t\r \u00a0but\t\r \u00a0extends\t\r \u00a0far\t\r \u00a0beyond\t\r \u00a0its\t\r \u00a0most\t\r \u00a0noted\t\r \u00a0signaling\t\r \u00a0role\t\r \u00a0in\t\r \u00a0preventing\t\r \u00a0apoptosis.\t\r \u00a0Our\t\r \u00a0data\t\r \u00a0provide\t\r \u00a0richly\t\r \u00a0detailed,\t\r \u00a0mechanistic\t\r \u00a0insight\t\r \u00a0into\t\r \u00a0several\t\r \u00a0phenomena\t\r \u00a0associated\t\r \u00a0with\t\r \u00a0Salmonella\t\r \u00a0invasion\t\r \u00a0(Figure\t\r \u00a04.6):\t\r \u00a0SopB\t\r \u00a0up-\u00ad\u2010regulates\t\r \u00a0Akt\u2019s\t\r \u00a0phosphorylation\t\r \u00a0of\t\r \u00a0an\t\r \u00a0inhibitory\t\r \u00a0site\t\r \u00a0(Ser-\u00ad\u201099)\t\r \u00a0in\t\r \u00a0the\t\r \u00a0pro-\u00ad\u2010apoptotic\t\r \u00a0protein\t\r \u00a0BAD,\t\r \u00a0which\t\r \u00a0supports\t\r \u00a0the\t\r \u00a0notion\t\r \u00a0that\t\r \u00a0SopB\t\r \u00a0down-\u00ad\u2010regulates\t\r \u00a0apoptosis\t\r \u00a0through\t\r \u00a0sequestration\t\r \u00a0of\t\r \u00a0BAD(165,\t\r \u00a0521).\t\r \u00a0Likewise,\t\r \u00a0during\t\r \u00a0infection\t\r \u00a0Rac1\t\r \u00a0 \t\r \u00a0\t\r \u00a0 134\t\r \u00a0 activation\/deactivation\t\r \u00a0is\t\r \u00a0controlled\t\r \u00a0by\t\r \u00a0SopE\/E2\t\r \u00a0and\t\r \u00a0SptP\t\r \u00a0to\t\r \u00a0regulate\t\r \u00a0membrane\t\r \u00a0ruffling\t\r \u00a0for\t\r \u00a0internalization\t\r \u00a0of\t\r \u00a0the\t\r \u00a0bacteria(45).\t\r \u00a0Intriguingly,\t\r \u00a0we\t\r \u00a0observe\t\r \u00a0an\t\r \u00a0inhibitory\t\r \u00a0Akt\t\r \u00a0phosphorylation\t\r \u00a0site\t\r \u00a0within\t\r \u00a0Rac1\t\r \u00a0that\t\r \u00a0is\t\r \u00a0regulated\t\r \u00a0by\t\r \u00a0SopB.\t\r \u00a0Since\t\r \u00a0Rac1\t\r \u00a0can\t\r \u00a0shut\t\r \u00a0down\t\r \u00a0the\t\r \u00a0actin\t\r \u00a0depolymerizing\t\r \u00a0activity\t\r \u00a0of\t\r \u00a0cofilin\t\r \u00a0through\t\r \u00a0LIMK\t\r \u00a0phosphorylation,\t\r \u00a0SopB\t\r \u00a0may\t\r \u00a0preserve\t\r \u00a0cofilin\t\r \u00a0activity\t\r \u00a0through\t\r \u00a0Rac1\t\r \u00a0inactivation\t\r \u00a0to\t\r \u00a0exert\t\r \u00a0an\t\r \u00a0effect\t\r \u00a0on\t\r \u00a0actin\t\r \u00a0similar\t\r \u00a0to\t\r \u00a0SptP(522,\t\r \u00a0523).\t\r \u00a0This\t\r \u00a0is\t\r \u00a0inconsistent\t\r \u00a0with\t\r \u00a0a\t\r \u00a0report\t\r \u00a0from\t\r \u00a0Patel\t\r \u00a0et\t\r \u00a0al.\t\r \u00a0(45)\t\r \u00a0showing\t\r \u00a0similar\t\r \u00a0levels\t\r \u00a0of\t\r \u00a0Rac1\t\r \u00a0activity\t\r \u00a020\t\r \u00a0min\t\r \u00a0post\t\r \u00a0infection\t\r \u00a0with\t\r \u00a0WT\t\r \u00a0vs.\t\r \u00a0\u2206sopB\t\r \u00a0 S.\t\r \u00a0Typhimurium.\t\r \u00a0However,\t\r \u00a0similar\t\r \u00a0to\t\r \u00a0SptP,\t\r \u00a0SopB\t\r \u00a0has\t\r \u00a0a\t\r \u00a0longer\t\r \u00a0half\t\r \u00a0life\t\r \u00a0in\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0as\t\r \u00a0compared\t\r \u00a0to\t\r \u00a0SopE,\t\r \u00a0indicating\t\r \u00a0that\t\r \u00a0this\t\r \u00a0effect\t\r \u00a0is\t\r \u00a0most\t\r \u00a0significant\t\r \u00a0at\t\r \u00a0later\t\r \u00a0times\t\r \u00a0post\t\r \u00a0infection(196,\t\r \u00a0197).\t\r \u00a0Also,\t\r \u00a0given\t\r \u00a0the\t\r \u00a0potential\t\r \u00a0redundancy\t\r \u00a0in\t\r \u00a0SopB\/SptP\t\r \u00a0function,\t\r \u00a0this\t\r \u00a0discrepancy\t\r \u00a0needs\t\r \u00a0to\t\r \u00a0be\t\r \u00a0addressed\t\r \u00a0using\t\r \u00a0a\t\r \u00a0\u2206sptP\t\r \u00a0background.\t\r \u00a0Lastly,\t\r \u00a0VAMP8\t\r \u00a0is\t\r \u00a0recruited\t\r \u00a0to\t\r \u00a0Salmonella-\u00ad\u2010induced\t\r \u00a0membrane\t\r \u00a0ruffles\t\r \u00a0where\t\r \u00a0it\t\r \u00a0promotes\t\r \u00a0invasion,\t\r \u00a0likely\t\r \u00a0by\t\r \u00a0providing\t\r \u00a0membranes\t\r \u00a0from\t\r \u00a0endocytic\t\r \u00a0compartments(50).\t\r \u00a0However,\t\r \u00a0the\t\r \u00a0t-\u00ad\u2010SNARE\t\r \u00a0target\t\r \u00a0of\t\r \u00a0VAMP8\t\r \u00a0during\t\r \u00a0Salmonella\t\r \u00a0invasion\t\r \u00a0was\t\r \u00a0previously\t\r \u00a0unknown.\t\r \u00a0Here\t\r \u00a0we\t\r \u00a0report\t\r \u00a0SopB\t\r \u00a0dependent\t\r \u00a0phosphorylation\t\r \u00a0of\t\r \u00a0STX7\t\r \u00a0at\t\r \u00a0two\t\r \u00a0sites\t\r \u00a0known\t\r \u00a0to\t\r \u00a0promote\t\r \u00a0complex\t\r \u00a0formation\t\r \u00a0with\t\r \u00a0VAMP8.\t\r \u00a0Thus,\t\r \u00a0STX7\t\r \u00a0is\t\r \u00a0likely\t\r \u00a0the\t\r \u00a0t-\u00ad\u2010SNARE\t\r \u00a0target\t\r \u00a0of\t\r \u00a0VAMP8\t\r \u00a0during\t\r \u00a0formation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV.\t\r \u00a0The\t\r \u00a0phosphoproteomic\t\r \u00a0analysis\t\r \u00a0presented\t\r \u00a0here\t\r \u00a0provides,\t\r \u00a0for\t\r \u00a0the\t\r \u00a0first\t\r \u00a0time,\t\r \u00a0a\t\r \u00a0comprehensive\t\r \u00a0picture\t\r \u00a0of\t\r \u00a0how\t\r \u00a0signaling\t\r \u00a0in\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0responds\t\r \u00a0to\t\r \u00a0a\t\r \u00a0bacterial\t\r \u00a0pathogen.\t\r \u00a0Extremely\t\r \u00a0low\t\r \u00a0biological\t\r \u00a0variability\t\r \u00a0(!CV\t\r \u00a0<\t\r \u00a015%)\t\r \u00a0and\t\r \u00a0rigorous\t\r \u00a0statistical\t\r \u00a0limits\t\r \u00a0mean\t\r \u00a0that\t\r \u00a0the\t\r \u00a0data\t\r \u00a0presented\t\r \u00a0here\t\r \u00a0is\t\r \u00a0a\t\r \u00a0powerful,\t\r \u00a0reliable\t\r \u00a0resource\t\r \u00a0for\t\r \u00a0generating\t\r \u00a0novel\t\r \u00a0testable\t\r \u00a0hypotheses\t\r \u00a0about\t\r \u00a0Salmonella\u2019s\t\r \u00a0actions.\t\r \u00a0Furthermore,\t\r \u00a0by\t\r \u00a0using\t\r \u00a0a\t\r \u00a0\u2206sopB\t\r \u00a0strain\t\r \u00a0to\t\r \u00a0attribute\t\r \u00a0specific\t\r \u00a0events\t\r \u00a0to\t\r \u00a0a\t\r \u00a0particular\t\r \u00a0effector,\t\r \u00a0we\t\r \u00a0have\t\r \u00a0 \t\r \u00a0\t\r \u00a0 135\t\r \u00a0 established\t\r \u00a0connections\t\r \u00a0between\t\r \u00a0previously\t\r \u00a0orphaned\t\r \u00a0observations\t\r \u00a0of\t\r \u00a0Salmonella\u2019s\t\r \u00a0impact\t\r \u00a0on\t\r \u00a0its\t\r \u00a0host.\t\r \u00a0These\t\r \u00a0data\t\r \u00a0provide\t\r \u00a0a\t\r \u00a0systems-\u00ad\u2010level\t\r \u00a0view\t\r \u00a0of\t\r \u00a0a\t\r \u00a0signaling\t\r \u00a0system\t\r \u00a0other\t\r \u00a0than\t\r \u00a0canonical\t\r \u00a0receptor-\u00ad\u2010mediated\t\r \u00a0cascades\t\r \u00a0and,\t\r \u00a0given\t\r \u00a0the\t\r \u00a0central\t\r \u00a0importance\t\r \u00a0of\t\r \u00a0bacterial-\u00ad\u2010induced\t\r \u00a0inflammation,\t\r \u00a0they\t\r \u00a0are\t\r \u00a0a\t\r \u00a0valuable\t\r \u00a0resource\t\r \u00a0for\t\r \u00a0understanding\t\r \u00a0host-\u00ad\u2010pathogen\t\r \u00a0interactions.\t\r \u00a0 Figure\t\r \u00a04.6\t\r \u00a0Activity\t\r \u00a0flow\t\r \u00a0diagram\t\r \u00a0of\t\r \u00a0phosphorylation\t\r \u00a0based\t\r \u00a0signaling\t\r \u00a0cascades\t\r \u00a0initiated\t\r \u00a0by\t\r \u00a0 T3SS-\u00ad\u20101\t\r \u00a0effectors\t\r \u00a0 \t\r \u00a0 Salmonella\t\r \u00a0effectors\t\r \u00a0are\t\r \u00a0shown\t\r \u00a0in\t\r \u00a0blue,\t\r \u00a0and\t\r \u00a0proteins\t\r \u00a0phosphorylated\t\r \u00a0during\t\r \u00a0infection\t\r \u00a0are\t\r \u00a0shown\t\r \u00a0in\t\r \u00a0red.\t\r \u00a0Biological\t\r \u00a0activities,\t\r \u00a0typically\t\r \u00a0labeled\t\r \u00a0with\t\r \u00a0the\t\r \u00a0gene\t\r \u00a0name,\t\r \u00a0are\t\r \u00a0shown\t\r \u00a0in\t\r \u00a0boxes,\t\r \u00a0and\t\r \u00a0the\t\r \u00a0influences\t\r \u00a0of\t\r \u00a0each\t\r \u00a0biological\t\r \u00a0activity\t\r \u00a0are\t\r \u00a0indicated\t\r \u00a0by\t\r \u00a0arcs\t\r \u00a0(lines)\t\r \u00a0directed\t\r \u00a0at\t\r \u00a0subsequent\t\r \u00a0biological\t\r \u00a0activities\t\r \u00a0or\t\r \u00a0at\t\r \u00a0phenotypes\t\r \u00a0(hexagonal\t\r \u00a0boxes).\t\r \u00a0Positive\t\r \u00a0influences\t\r \u00a0are\t\r \u00a0indicated\t\r \u00a0by\t\r \u00a0arrowheads\t\r \u00a0on\t\r \u00a0an\t\r \u00a0arc,\t\r \u00a0negative\t\r \u00a0influences\t\r \u00a0by\t\r \u00a0a\t\r \u00a0short\t\r \u00a0perpendicular\t\r \u00a0line\t\r \u00a0at\t\r \u00a0the\t\r \u00a0end\t\r \u00a0of\t\r \u00a0an\t\r \u00a0arc,\t\r \u00a0and\t\r \u00a0unknown\t\r \u00a0influence\t\r \u00a0is\t\r \u00a0shown\t\r \u00a0by\t\r \u00a0a\t\r \u00a0diamond\t\r \u00a0shape\t\r \u00a0at\t\r \u00a0the\t\r \u00a0end\t\r \u00a0of\t\r \u00a0an\t\r \u00a0arc\t\r \u00a0(i.e.\t\r \u00a0SspH1).\t\r \u00a0A\t\r \u00a0necessary\t\r \u00a0influence\t\r \u00a0(one\t\r \u00a0required\t\r \u00a0for\t\r \u00a0the\t\r \u00a0subsequent\t\r \u00a0biological\t\r \u00a0activity)\t\r \u00a0is\t\r \u00a0indicated\t\r \u00a0by\t\r \u00a0a\t\r \u00a0perpendicular\t\r \u00a0line\t\r \u00a0followed\t\r \u00a0by\t\r \u00a0an\t\r \u00a0arrowhead\t\r \u00a0(i.e.\t\r \u00a0IL-\u00ad\u20108).\t\r \u00a0Modifiers\t\r \u00a0added\t\r \u00a0to\t\r \u00a0arcs\t\r \u00a0include\t\r \u00a0Boolean\t\r \u00a0terms\t\r \u00a0and\t\r \u00a0the\t\r \u00a0greek\t\r \u00a0letter\t\r \u00a0'tau',\t\r \u00a0which\t\r \u00a0indicates\t\r \u00a0that\t\r \u00a0the\t\r \u00a0biological\t\r \u00a0activity\t\r \u00a0is\t\r \u00a0delayed.\t\r \u00a0This\t\r \u00a0diagram\t\r \u00a0is\t\r \u00a0designed\t\r \u00a0to\t\r \u00a0conform\t\r \u00a0to\t\r \u00a0the\t\r \u00a0systems\t\r \u00a0biology\t\r \u00a0graphic\t\r \u00a0notation(195).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 136\t\r \u00a0 5\t\r \u00a0Conclusion\t\r \u00a0 The\t\r \u00a0research\t\r \u00a0presented\t\r \u00a0in\t\r \u00a0this\t\r \u00a0thesis\t\r \u00a0has\t\r \u00a0focused\t\r \u00a0on\t\r \u00a0the\t\r \u00a0identification\t\r \u00a0of\t\r \u00a0novel\t\r \u00a0host\t\r \u00a0targets\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0effector\t\r \u00a0SopB.\t\r \u00a0This\t\r \u00a0has\t\r \u00a0been\t\r \u00a0done\t\r \u00a0using\t\r \u00a0primarily\t\r \u00a0quantitative\t\r \u00a0mass\t\r \u00a0spectrometry-\u00ad\u2010based\t\r \u00a0proteomics\t\r \u00a0and\t\r \u00a0bioinformatics\t\r \u00a0techniques.\t\r \u00a0SILAC\t\r \u00a0immunoprecipitations\t\r \u00a0following\t\r \u00a0exogenous\t\r \u00a0SopB\t\r \u00a0expression\t\r \u00a0in\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0identified\t\r \u00a0Cdc42\t\r \u00a0as\t\r \u00a0a\t\r \u00a0novel\t\r \u00a0direct\t\r \u00a0binding\t\r \u00a0partner\t\r \u00a0to\t\r \u00a0this\t\r \u00a0effector\t\r \u00a0(Chapter\t\r \u00a02).\t\r \u00a0The\t\r \u00a0binding\t\r \u00a0site\t\r \u00a0within\t\r \u00a0SopB\t\r \u00a0was\t\r \u00a0localized,\t\r \u00a0and\t\r \u00a0SopB\t\r \u00a0and\t\r \u00a0Cdc42\t\r \u00a0were\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0colocalize\t\r \u00a0in\t\r \u00a0membrane\t\r \u00a0ruffles\t\r \u00a0at\t\r \u00a0the\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0surface.\t\r \u00a0To\t\r \u00a0globally\t\r \u00a0analyze\t\r \u00a0dynamics\t\r \u00a0in\t\r \u00a0host\t\r \u00a0protein\t\r \u00a0phosphorylation\t\r \u00a0during\t\r \u00a0Salmonella\t\r \u00a0infection,\t\r \u00a0a\t\r \u00a0phosphoproteomics\t\r \u00a0method\t\r \u00a0employing\t\r \u00a0heat\t\r \u00a0and\t\r \u00a0detergent\t\r \u00a0based\t\r \u00a0phosphatase\t\r \u00a0inactivation,\t\r \u00a0MOC,\t\r \u00a0IEF\t\r \u00a0and\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0was\t\r \u00a0developed\t\r \u00a0(Chapter\t\r \u00a03).\t\r \u00a0Applying\t\r \u00a0this\t\r \u00a0technique\t\r \u00a0along\t\r \u00a0with\t\r \u00a0SILAC\t\r \u00a0quantitation\t\r \u00a0for\t\r \u00a020\t\r \u00a0min\t\r \u00a0post\t\r \u00a0infection\t\r \u00a0by\t\r \u00a0Salmonella\t\r \u00a0generated\t\r \u00a0thousands\t\r \u00a0of\t\r \u00a0dynamic\t\r \u00a0host\t\r \u00a0phosphopeptide\t\r \u00a0profiles\t\r \u00a0(Chapter\t\r \u00a04).\t\r \u00a0Kinase\t\r \u00a0prediction\t\r \u00a0upstream\t\r \u00a0of\t\r \u00a0these\t\r \u00a0sites\t\r \u00a0identified\t\r \u00a0Akt\t\r \u00a0and\t\r \u00a0PKC\t\r \u00a0as\t\r \u00a0master\t\r \u00a0regulators\t\r \u00a0targeted\t\r \u00a0during\t\r \u00a0infection.\t\r \u00a0Comparing\t\r \u00a0phosphorylation\t\r \u00a0dynamics\t\r \u00a0following\t\r \u00a0WT\t\r \u00a0and\t\r \u00a0\u0394sopB\t\r \u00a0infection\t\r \u00a0revealed\t\r \u00a0that\t\r \u00a0up\t\r \u00a0to\t\r \u00a0half\t\r \u00a0of\t\r \u00a0the\t\r \u00a0phosphosites\t\r \u00a0regulated\t\r \u00a0during\t\r \u00a0infection\t\r \u00a0are\t\r \u00a0SopB\t\r \u00a0dependent,\t\r \u00a0and\t\r \u00a0several\t\r \u00a0novel\t\r \u00a0host\t\r \u00a0proteins\t\r \u00a0targeted\t\r \u00a0downstream\t\r \u00a0of\t\r \u00a0this\t\r \u00a0effector\t\r \u00a0were\t\r \u00a0identified.\t\r \u00a0\t\r \u00a0 This\t\r \u00a0work\t\r \u00a0has\t\r \u00a0made\t\r \u00a0several\t\r \u00a0contributions\t\r \u00a0to\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0field\t\r \u00a0and,\t\r \u00a0more\t\r \u00a0generally,\t\r \u00a0to\t\r \u00a0cell\t\r \u00a0signaling\t\r \u00a0and\t\r \u00a0host-\u00ad\u2010pathogen\t\r \u00a0research.\t\r \u00a0Most\t\r \u00a0specifically,\t\r \u00a0several\t\r \u00a0proteins\t\r \u00a0targeted\t\r \u00a0downstream\t\r \u00a0of\t\r \u00a0the\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0effector\t\r \u00a0SopB\t\r \u00a0have\t\r \u00a0been\t\r \u00a0identified.\t\r \u00a0SopB\t\r \u00a0is\t\r \u00a0known\t\r \u00a0to\t\r \u00a0regulate\t\r \u00a0several\t\r \u00a0processes\t\r \u00a0within\t\r \u00a0host\t\r \u00a0cells,\t\r \u00a0including\t\r \u00a0the\t\r \u00a0induction\t\r \u00a0of\t\r \u00a0innate\t\r \u00a0immune\t\r \u00a0signaling\t\r \u00a0cascades,\t\r \u00a0membrane\t\r \u00a0ruffling\t\r \u00a0for\t\r \u00a0bacterial\t\r \u00a0 \t\r \u00a0\t\r \u00a0 137\t\r \u00a0 internalization,\t\r \u00a0and\t\r \u00a0maturation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SCV,\t\r \u00a0all\t\r \u00a0of\t\r \u00a0which\t\r \u00a0are\t\r \u00a0central\t\r \u00a0to\t\r \u00a0pathogenesis\t\r \u00a0and\t\r \u00a0disease(45,\t\r \u00a058).\t\r \u00a0The\t\r \u00a0host\t\r \u00a0targets\t\r \u00a0identified\t\r \u00a0here\t\r \u00a0have\t\r \u00a0greatly\t\r \u00a0enhanced\t\r \u00a0our\t\r \u00a0understanding\t\r \u00a0of\t\r \u00a0the\t\r \u00a0molecular\t\r \u00a0detail\t\r \u00a0through\t\r \u00a0which\t\r \u00a0SopB\t\r \u00a0regulates\t\r \u00a0these\t\r \u00a0processes\t\r \u00a0(i.e.\t\r \u00a0direct\t\r \u00a0interaction\t\r \u00a0with\t\r \u00a0Cdc42,\t\r \u00a0activation\t\r \u00a0of\t\r \u00a0STX7\t\r \u00a0and\t\r \u00a0inactivation\t\r \u00a0of\t\r \u00a0BAD),\t\r \u00a0while\t\r \u00a0also\t\r \u00a0identifying\t\r \u00a0novel\t\r \u00a0host\t\r \u00a0activities\t\r \u00a0manipulated\t\r \u00a0by\t\r \u00a0this\t\r \u00a0effector\t\r \u00a0(i.e.\t\r \u00a0inactivation\t\r \u00a0of\t\r \u00a0RAC1).\t\r \u00a0Furthermore,\t\r \u00a0phosphoproteomic\t\r \u00a0analysis\t\r \u00a0has\t\r \u00a0provided,\t\r \u00a0for\t\r \u00a0the\t\r \u00a0first\t\r \u00a0time,\t\r \u00a0a\t\r \u00a0global\t\r \u00a0view\t\r \u00a0of\t\r \u00a0protein\t\r \u00a0phosphorylation\t\r \u00a0cascades\t\r \u00a0induced\t\r \u00a0in\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0during\t\r \u00a0bacterial\t\r \u00a0infection.\t\r \u00a0This\t\r \u00a0data\t\r \u00a0is\t\r \u00a0expected\t\r \u00a0to\t\r \u00a0be\t\r \u00a0an\t\r \u00a0excellent\t\r \u00a0resource\t\r \u00a0for\t\r \u00a0opening\t\r \u00a0new\t\r \u00a0avenues\t\r \u00a0of\t\r \u00a0research\t\r \u00a0pertaining\t\r \u00a0to\t\r \u00a0Salmonella,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0other\t\r \u00a0closely\t\r \u00a0related\t\r \u00a0pathogens.\t\r \u00a0In\t\r \u00a0addition,\t\r \u00a0signaling\t\r \u00a0downstream\t\r \u00a0of\t\r \u00a0bacterial\t\r \u00a0effectors\t\r \u00a0represents\t\r \u00a0a\t\r \u00a0unique\t\r \u00a0signaling\t\r \u00a0mechanism\t\r \u00a0with\t\r \u00a0distinct\t\r \u00a0players\t\r \u00a0and\t\r \u00a0dynamics\t\r \u00a0to\t\r \u00a0canonical\t\r \u00a0ligand-\u00ad\u2010receptor\t\r \u00a0induced\t\r \u00a0cascades.\t\r \u00a0This\t\r \u00a0data\t\r \u00a0can\t\r \u00a0be\t\r \u00a0used\t\r \u00a0as\t\r \u00a0a\t\r \u00a0model\t\r \u00a0within\t\r \u00a0the\t\r \u00a0cell\t\r \u00a0signaling\t\r \u00a0field\t\r \u00a0at\t\r \u00a0large.\t\r \u00a0Finally,\t\r \u00a0while\t\r \u00a0employed\t\r \u00a0here\t\r \u00a0to\t\r \u00a0identify\t\r \u00a0novel\t\r \u00a0host\t\r \u00a0targets\t\r \u00a0of\t\r \u00a0a\t\r \u00a0single\t\r \u00a0Salmonella\t\r \u00a0effector,\t\r \u00a0these\t\r \u00a0studies\t\r \u00a0represent\t\r \u00a0the\t\r \u00a0first\t\r \u00a0application\t\r \u00a0of\t\r \u00a0SILAC\t\r \u00a0immunoprecipitations\t\r \u00a0and\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0based\t\r \u00a0phosphoproteomics\t\r \u00a0to\t\r \u00a0study\t\r \u00a0bacterial\t\r \u00a0pathogenesis.\t\r \u00a0Thus,\t\r \u00a0it\t\r \u00a0is\t\r \u00a0expected\t\r \u00a0that\t\r \u00a0the\t\r \u00a0techniques\t\r \u00a0developed\t\r \u00a0here\t\r \u00a0will\t\r \u00a0be\t\r \u00a0widely\t\r \u00a0applied\t\r \u00a0to\t\r \u00a0study\t\r \u00a0other\t\r \u00a0Salmonella\t\r \u00a0effectors,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0those\t\r \u00a0from\t\r \u00a0other\t\r \u00a0pathogens.\t\r \u00a0\t\r \u00a0 Looking\t\r \u00a0back\t\r \u00a0on\t\r \u00a0research\t\r \u00a0done\t\r \u00a0within\t\r \u00a0this\t\r \u00a0thesis,\t\r \u00a0it\t\r \u00a0is\t\r \u00a0evident\t\r \u00a0that\t\r \u00a0several\t\r \u00a0experiments\t\r \u00a0could\t\r \u00a0be\t\r \u00a0conducted\t\r \u00a0to\t\r \u00a0both\t\r \u00a0improve\t\r \u00a0and\t\r \u00a0further\t\r \u00a0characterize\t\r \u00a0the\t\r \u00a0findings\t\r \u00a0presented\t\r \u00a0within.\t\r \u00a0To\t\r \u00a0identify\t\r \u00a0host-\u00ad\u2010binding\t\r \u00a0partners\t\r \u00a0to\t\r \u00a0SopB,\t\r \u00a0a\t\r \u00a0tagged-\u00ad\u2010SopB\t\r \u00a0construct\t\r \u00a0was\t\r \u00a0exogenously\t\r \u00a0expressed\t\r \u00a0in\t\r \u00a0HEK293\t\r \u00a0cells\t\r \u00a0and\t\r \u00a0immunoprecipitated\t\r \u00a0for\t\r \u00a0LC-\u00ad\u2010MSn\t\r \u00a0analysis.\t\r \u00a0However,\t\r \u00a0it\t\r \u00a0would\t\r \u00a0be\t\r \u00a0much\t\r \u00a0more\t\r \u00a0informative\t\r \u00a0to\t\r \u00a0deliver\t\r \u00a0SopB\t\r \u00a0into\t\r \u00a0 \t\r \u00a0\t\r \u00a0 138\t\r \u00a0 host\t\r \u00a0cells\t\r \u00a0through\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0during\t\r \u00a0infection.\t\r \u00a0It\t\r \u00a0is\t\r \u00a0widely\t\r \u00a0believed\t\r \u00a0within\t\r \u00a0the\t\r \u00a0 Salmonella\t\r \u00a0community\t\r \u00a0that\t\r \u00a0effectors\t\r \u00a0are\t\r \u00a0delivered\t\r \u00a0into\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0in\t\r \u00a0very\t\r \u00a0low\t\r \u00a0amounts,\t\r \u00a0and\t\r \u00a0this\t\r \u00a0prompted\t\r \u00a0us\t\r \u00a0to\t\r \u00a0use\t\r \u00a0exogenous\t\r \u00a0expression\t\r \u00a0to\t\r \u00a0obtain\t\r \u00a0protein\t\r \u00a0quantities\t\r \u00a0sufficient\t\r \u00a0for\t\r \u00a0LC-\u00ad\u2010MSn.\t\r \u00a0However,\t\r \u00a0we\t\r \u00a0subsequently\t\r \u00a0observed\t\r \u00a0that\t\r \u00a0at\t\r \u00a0least\t\r \u00a0as\t\r \u00a0much\t\r \u00a0SopB\t\r \u00a0is\t\r \u00a0delivered\t\r \u00a0by\t\r \u00a0bacteria\t\r \u00a0as\t\r \u00a0can\t\r \u00a0be\t\r \u00a0obtained\t\r \u00a0through\t\r \u00a0exogenous\t\r \u00a0expression.\t\r \u00a0As\t\r \u00a0SopB\t\r \u00a0persists\t\r \u00a0in\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0for\t\r \u00a0several\t\r \u00a0hours\t\r \u00a0following\t\r \u00a0infection\t\r \u00a0and\t\r \u00a0relocalizes\t\r \u00a0from\t\r \u00a0the\t\r \u00a0cell\t\r \u00a0membrane\t\r \u00a0to\t\r \u00a0the\t\r \u00a0SCV,\t\r \u00a0this\t\r \u00a0would\t\r \u00a0have\t\r \u00a0enabled\t\r \u00a0us\t\r \u00a0to\t\r \u00a0employ\t\r \u00a0SILAC\t\r \u00a0immunoprecipitations\t\r \u00a0at\t\r \u00a0various\t\r \u00a0times\t\r \u00a0post\t\r \u00a0infection\t\r \u00a0and\t\r \u00a0potentially\t\r \u00a0identify\t\r \u00a0binding\t\r \u00a0partners\t\r \u00a0specific\t\r \u00a0to\t\r \u00a0particular\t\r \u00a0stages\t\r \u00a0of\t\r \u00a0SCV\t\r \u00a0maturation(197).\t\r \u00a0Also,\t\r \u00a0Cdc42\t\r \u00a0activation\t\r \u00a0is\t\r \u00a0known\t\r \u00a0to\t\r \u00a0occur\t\r \u00a0downstream\t\r \u00a0of\t\r \u00a0both\t\r \u00a0SopB\t\r \u00a0and\t\r \u00a0SopE\/E2\t\r \u00a0during\t\r \u00a0infection,\t\r \u00a0but\t\r \u00a0it\t\r \u00a0was\t\r \u00a0not\t\r \u00a0activated\t\r \u00a0following\t\r \u00a0exogenous\t\r \u00a0expression\t\r \u00a0of\t\r \u00a0SopB(45).\t\r \u00a0While\t\r \u00a0Cdc42\t\r \u00a0is\t\r \u00a0the\t\r \u00a0only\t\r \u00a0protein\t\r \u00a0identified\t\r \u00a0to\t\r \u00a0interact\t\r \u00a0with\t\r \u00a0SopB\t\r \u00a0in\t\r \u00a0the\t\r \u00a0work\t\r \u00a0presented\t\r \u00a0here,\t\r \u00a0it\t\r \u00a0is\t\r \u00a0likely\t\r \u00a0that\t\r \u00a0another\t\r \u00a0Salmonella\t\r \u00a0protein\t\r \u00a0acts\t\r \u00a0alongside\t\r \u00a0SopB\t\r \u00a0for\t\r \u00a0Cdc42\t\r \u00a0activation.\t\r \u00a0Thus,\t\r \u00a0it\t\r \u00a0would\t\r \u00a0be\t\r \u00a0beneficial\t\r \u00a0to\t\r \u00a0repeat\t\r \u00a0the\t\r \u00a0SILAC\t\r \u00a0immunoprecipitation\t\r \u00a0experiments\t\r \u00a0using\t\r \u00a0bacterial\t\r \u00a0delivered\t\r \u00a0SopB\t\r \u00a0and\t\r \u00a0assay\t\r \u00a0for\t\r \u00a0binding\t\r \u00a0partners\t\r \u00a0at\t\r \u00a0various\t\r \u00a0times\t\r \u00a0post\t\r \u00a0infection.\t\r \u00a0\t\r \u00a0 Phosphoproteomic\t\r \u00a0analysis\t\r \u00a0provided\t\r \u00a0temporal\t\r \u00a0profiles\t\r \u00a0of\t\r \u00a0more\t\r \u00a0than\t\r \u00a08000\t\r \u00a0phosphopeptides\t\r \u00a0during\t\r \u00a0the\t\r \u00a0initial\t\r \u00a0stages\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0infection.\t\r \u00a0While\t\r \u00a0this\t\r \u00a0is\t\r \u00a0a\t\r \u00a0large\t\r \u00a0dataset\t\r \u00a0and\t\r \u00a0provides\t\r \u00a0several\t\r \u00a0new\t\r \u00a0insights\t\r \u00a0into\t\r \u00a0Salmonella\t\r \u00a0pathogenesis,\t\r \u00a0it\t\r \u00a0is\t\r \u00a0unlikely\t\r \u00a0that\t\r \u00a0the\t\r \u00a0complete\t\r \u00a0HeLa\t\r \u00a0cell\t\r \u00a0phosphoproteome\t\r \u00a0is\t\r \u00a0represented.\t\r \u00a0The\t\r \u00a0number\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0identified\t\r \u00a0in\t\r \u00a0this\t\r \u00a0study\t\r \u00a0was\t\r \u00a0likely\t\r \u00a0limited\t\r \u00a0for\t\r \u00a0a\t\r \u00a0number\t\r \u00a0of\t\r \u00a0reasons.\t\r \u00a0The\t\r \u00a0first\t\r \u00a0is\t\r \u00a0due\t\r \u00a0to\t\r \u00a0the\t\r \u00a0maximum\t\r \u00a0capacity\t\r \u00a0of\t\r \u00a0the\t\r \u00a0MicroRotofor,\t\r \u00a0which\t\r \u00a0is\t\r \u00a0approximately\t\r \u00a030\t\r \u00a0mg\t\r \u00a0(Chapter\t\r \u00a03).\t\r \u00a0Following\t\r \u00a0phosphopeptide\t\r \u00a0enrichment\t\r \u00a0within\t\r \u00a0 \t\r \u00a0\t\r \u00a0 139\t\r \u00a0 each\t\r \u00a0IEF\t\r \u00a0fraction,\t\r \u00a0the\t\r \u00a0material\t\r \u00a0loaded\t\r \u00a0onto\t\r \u00a0the\t\r \u00a0mass\t\r \u00a0spectrometer\t\r \u00a0was\t\r \u00a0not\t\r \u00a0saturating\t\r \u00a0the\t\r \u00a0instrument,\t\r \u00a0suggesting\t\r \u00a0that\t\r \u00a0a\t\r \u00a0pre-\u00ad\u2010fractionation\t\r \u00a0technique\t\r \u00a0capable\t\r \u00a0of\t\r \u00a0higher\t\r \u00a0loading\t\r \u00a0capacities\t\r \u00a0(i.e.\t\r \u00a0BIORAD\t\r \u00a0Rotofor)\t\r \u00a0and\t\r \u00a0scaling\t\r \u00a0up\t\r \u00a0the\t\r \u00a0size\t\r \u00a0of\t\r \u00a0each\t\r \u00a0experiment\t\r \u00a0would\t\r \u00a0be\t\r \u00a0beneficial.\t\r \u00a0Secondly,\t\r \u00a0the\t\r \u00a0various\t\r \u00a0isotopologs\t\r \u00a0of\t\r \u00a0SILAC\t\r \u00a0labeled\t\r \u00a0peptides\t\r \u00a0complicate\t\r \u00a0MS\t\r \u00a0spectra\t\r \u00a0and\t\r \u00a0can\t\r \u00a0result\t\r \u00a0in\t\r \u00a0fewer\t\r \u00a0protein\t\r \u00a0identifications\t\r \u00a0as\t\r \u00a0compared\t\r \u00a0to\t\r \u00a0unlabeled\t\r \u00a0samples.\t\r \u00a0An\t\r \u00a0alternative\t\r \u00a0technique\t\r \u00a0is\t\r \u00a0iTRAQ,\t\r \u00a0where\t\r \u00a0all\t\r \u00a0labeled\t\r \u00a0forms\t\r \u00a0of\t\r \u00a0a\t\r \u00a0peptide\t\r \u00a0are\t\r \u00a0the\t\r \u00a0same\t\r \u00a0mass,\t\r \u00a0and\t\r \u00a0quantitation\t\r \u00a0is\t\r \u00a0achieved\t\r \u00a0in\t\r \u00a0higher\t\r \u00a0order\t\r \u00a0spectra(344).\t\r \u00a0iTRAQ\t\r \u00a0also\t\r \u00a0offers\t\r \u00a08\t\r \u00a0isotopic\t\r \u00a0labels\t\r \u00a0which\t\r \u00a0could\t\r \u00a0be\t\r \u00a0used\t\r \u00a0for\t\r \u00a0a\t\r \u00a0more\t\r \u00a0highly\t\r \u00a0resolved\t\r \u00a0or\t\r \u00a0longer\t\r \u00a0infection\t\r \u00a0time\t\r \u00a0course(345).\t\r \u00a0At\t\r \u00a0the\t\r \u00a0time\t\r \u00a0of\t\r \u00a0this\t\r \u00a0study,\t\r \u00a0a\t\r \u00a0high\t\r \u00a0end\t\r \u00a0mass\t\r \u00a0spectrometer\t\r \u00a0applicable\t\r \u00a0to\t\r \u00a0identifying\t\r \u00a0the\t\r \u00a0low\t\r \u00a0m\/z\t\r \u00a0reporter\t\r \u00a0ions\t\r \u00a0generated\t\r \u00a0by\t\r \u00a0the\t\r \u00a0iTRAQ\t\r \u00a0labels\t\r \u00a0was\t\r \u00a0not\t\r \u00a0available\t\r \u00a0in\t\r \u00a0Dr.\t\r \u00a0Foster\u2019s\t\r \u00a0research\t\r \u00a0group.\t\r \u00a0However,\t\r \u00a0such\t\r \u00a0an\t\r \u00a0instrument\t\r \u00a0(quadrupole\t\r \u00a0time-\u00ad\u2010of-\u00ad\u2010flight)\t\r \u00a0has\t\r \u00a0recently\t\r \u00a0been\t\r \u00a0acquired\t\r \u00a0and\t\r \u00a0could\t\r \u00a0also\t\r \u00a0be\t\r \u00a0used\t\r \u00a0to\t\r \u00a0validate\t\r \u00a0phosphosite\t\r \u00a0locations,\t\r \u00a0some\t\r \u00a0of\t\r \u00a0which\t\r \u00a0might\t\r \u00a0have\t\r \u00a0been\t\r \u00a0scrambled\t\r \u00a0during\t\r \u00a0slow\t\r \u00a0CID\t\r \u00a0within\t\r \u00a0the\t\r \u00a0ion\t\r \u00a0trap(425).\t\r \u00a0Finally,\t\r \u00a0as\t\r \u00a0with\t\r \u00a0all\t\r \u00a0proteomics\t\r \u00a0experiments,\t\r \u00a0the\t\r \u00a0number\t\r \u00a0of\t\r \u00a0protein\t\r \u00a0identifications\t\r \u00a0is\t\r \u00a0almost\t\r \u00a0always\t\r \u00a0limited\t\r \u00a0by\t\r \u00a0the\t\r \u00a0sensitivity\t\r \u00a0and\t\r \u00a0scan\t\r \u00a0speed\t\r \u00a0of\t\r \u00a0the\t\r \u00a0mass\t\r \u00a0spectrometer.\t\r \u00a0The\t\r \u00a0work\t\r \u00a0presented\t\r \u00a0here\t\r \u00a0utilized\t\r \u00a0an\t\r \u00a0LTQ-\u00ad\u2010Orbitrap\t\r \u00a0instrument(301).\t\r \u00a0However,\t\r \u00a0this\t\r \u00a0instrument\t\r \u00a0has\t\r \u00a0recently\t\r \u00a0been\t\r \u00a0upgraded\t\r \u00a0to\t\r \u00a0the\t\r \u00a0next\t\r \u00a0generation\t\r \u00a0LTQ-\u00ad\u2010Orbitrap\t\r \u00a0Velos\t\r \u00a0with\t\r \u00a0greatly\t\r \u00a0improved\t\r \u00a0sensitivity\t\r \u00a0and\t\r \u00a0speed,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0an\t\r \u00a0HCD\t\r \u00a0cell\t\r \u00a0which,\t\r \u00a0similar\t\r \u00a0to\t\r \u00a0CID\t\r \u00a0in\t\r \u00a0a\t\r \u00a0quadrupole,\t\r \u00a0does\t\r \u00a0not\t\r \u00a0allow\t\r \u00a0phosphosite\t\r \u00a0scrambling(355).\t\r \u00a0 \t\r \u00a0Based\t\r \u00a0upon\t\r \u00a0the\t\r \u00a0data\t\r \u00a0and\t\r \u00a0methods\t\r \u00a0presented\t\r \u00a0within\t\r \u00a0Chapters\t\r \u00a03\t\r \u00a0and\t\r \u00a04\t\r \u00a0of\t\r \u00a0this\t\r \u00a0thesis,\t\r \u00a0a\t\r \u00a0myriad\t\r \u00a0of\t\r \u00a0additional\t\r \u00a0phosphoproteomics\t\r \u00a0experiments\t\r \u00a0could\t\r \u00a0be\t\r \u00a0employed\t\r \u00a0to\t\r \u00a0further\t\r \u00a0study\t\r \u00a0Salmonella-\u00ad\u2010host\t\r \u00a0interactions.\t\r \u00a0In\t\r \u00a0addition\t\r \u00a0to\t\r \u00a0SopB,\t\r \u00a0the\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0 \t\r \u00a0\t\r \u00a0 140\t\r \u00a0 effectors\t\r \u00a0SopE\/E2\t\r \u00a0and\t\r \u00a0SipA\t\r \u00a0are\t\r \u00a0also\t\r \u00a0known\t\r \u00a0to\t\r \u00a0induce\t\r \u00a0phosphorylation\t\r \u00a0signaling\t\r \u00a0within\t\r \u00a0host\t\r \u00a0cells,\t\r \u00a0and\t\r \u00a0PKC,\t\r \u00a0which\t\r \u00a0has\t\r \u00a0been\t\r \u00a0shown\t\r \u00a0to\t\r \u00a0be\t\r \u00a0activated\t\r \u00a0downstream\t\r \u00a0of\t\r \u00a0SipA,\t\r \u00a0was\t\r \u00a0identified\t\r \u00a0as\t\r \u00a0a\t\r \u00a0global\t\r \u00a0host\t\r \u00a0regulator\t\r \u00a0during\t\r \u00a0WT\t\r \u00a0infection(45,\t\r \u00a0147).\t\r \u00a0Thus,\t\r \u00a0it\t\r \u00a0would\t\r \u00a0be\t\r \u00a0interesting\t\r \u00a0to\t\r \u00a0compare\t\r \u00a0phosphopeptide\t\r \u00a0dynamics\t\r \u00a0following\t\r \u00a0infection\t\r \u00a0by\t\r \u00a0WT\t\r \u00a0versus\t\r \u00a0\u0394sipA\t\r \u00a0and\t\r \u00a0\u0394sopE\/E2.\t\r \u00a0This\t\r \u00a0could\t\r \u00a0be\t\r \u00a0done\t\r \u00a0using\t\r \u00a0the\t\r \u00a0method\t\r \u00a0presented\t\r \u00a0in\t\r \u00a0Chapter\t\r \u00a03,\t\r \u00a0or\t\r \u00a0through\t\r \u00a0development\t\r \u00a0of\t\r \u00a0multiple\t\r \u00a0reaction\t\r \u00a0monitoring\t\r \u00a0(MRM)\t\r \u00a0assays(542).\t\r \u00a0In\t\r \u00a0the\t\r \u00a0later,\t\r \u00a0tryptic\t\r \u00a0peptides\t\r \u00a0containing\t\r \u00a0regulated\t\r \u00a0phosphosites\t\r \u00a0from\t\r \u00a0interesting\t\r \u00a0host\t\r \u00a0proteins\t\r \u00a0would\t\r \u00a0be\t\r \u00a0synthesized\t\r \u00a0in\t\r \u00a0an\t\r \u00a0isotopically\t\r \u00a0labeled\t\r \u00a0form.\t\r \u00a0These\t\r \u00a0synthetic\t\r \u00a0peptides\t\r \u00a0would\t\r \u00a0then\t\r \u00a0be\t\r \u00a0spiked\t\r \u00a0into\t\r \u00a0samples\t\r \u00a0derived\t\r \u00a0from\t\r \u00a0WT\t\r \u00a0infection\t\r \u00a0or\t\r \u00a0infection\t\r \u00a0by\t\r \u00a0a\t\r \u00a0deletion\t\r \u00a0mutant\t\r \u00a0strain\t\r \u00a0for\t\r \u00a0any\t\r \u00a0number\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0or\t\r \u00a0-\u00ad\u20102\t\r \u00a0effectors.\t\r \u00a0The\t\r \u00a0mass\t\r \u00a0spectrometer\t\r \u00a0(triple\t\r \u00a0quadrupole)\t\r \u00a0would\t\r \u00a0then\t\r \u00a0be\t\r \u00a0set\t\r \u00a0to\t\r \u00a0monitor\t\r \u00a0only\t\r \u00a0these\t\r \u00a0selected\t\r \u00a0peptides,\t\r \u00a0and\t\r \u00a0their\t\r \u00a0absolute\t\r \u00a0abundance\t\r \u00a0and\t\r \u00a0regulation\t\r \u00a0could\t\r \u00a0be\t\r \u00a0determined.\t\r \u00a0The\t\r \u00a0phosphoproteomic\t\r \u00a0data\t\r \u00a0presented\t\r \u00a0here\t\r \u00a0studied\t\r \u00a0only\t\r \u00a0the\t\r \u00a0initial\t\r \u00a020\t\r \u00a0min\t\r \u00a0post\t\r \u00a0infection\t\r \u00a0of\t\r \u00a0human\t\r \u00a0epithelial\t\r \u00a0cells\t\r \u00a0by\t\r \u00a0S.\t\r \u00a0Typhimurium.\t\r \u00a0It\t\r \u00a0would\t\r \u00a0be\t\r \u00a0interesting\t\r \u00a0to\t\r \u00a0extend\t\r \u00a0this\t\r \u00a0time\t\r \u00a0course\t\r \u00a0to\t\r \u00a0later\t\r \u00a0stages\t\r \u00a0of\t\r \u00a0infection\t\r \u00a0such\t\r \u00a0as\t\r \u00a0SCV\t\r \u00a0maturation\t\r \u00a0(20-\u00ad\u201060\t\r \u00a0min\t\r \u00a0post\t\r \u00a0infection)\t\r \u00a0and\t\r \u00a0during\t\r \u00a0Salmonella\t\r \u00a0replication\t\r \u00a0and\t\r \u00a0SIF\t\r \u00a0formation\t\r \u00a0(4-\u00ad\u20106\t\r \u00a0h\t\r \u00a0post\t\r \u00a0infection).\t\r \u00a0These\t\r \u00a0studies\t\r \u00a0could\t\r \u00a0also\t\r \u00a0employ\t\r \u00a0other\t\r \u00a0cell\t\r \u00a0types,\t\r \u00a0which\t\r \u00a0would\t\r \u00a0be\t\r \u00a0very\t\r \u00a0interesting\t\r \u00a0since\t\r \u00a0Salmonella\t\r \u00a0invasion\t\r \u00a0of\t\r \u00a0macrophages\t\r \u00a0is\t\r \u00a0known\t\r \u00a0to\t\r \u00a0induce\t\r \u00a0a\t\r \u00a0unique\t\r \u00a0form\t\r \u00a0of\t\r \u00a0cell\t\r \u00a0death\t\r \u00a0termed\t\r \u00a0pyroptosis(40).\t\r \u00a0Furthermore,\t\r \u00a0S.\t\r \u00a0Typhimurium\t\r \u00a0cause\t\r \u00a0gastroenteritis\t\r \u00a0in\t\r \u00a0humans\t\r \u00a0and\t\r \u00a0enteric\t\r \u00a0fever\t\r \u00a0in\t\r \u00a0mice\t\r \u00a0and\t\r \u00a0thus,\t\r \u00a0can\t\r \u00a0survive\t\r \u00a0and\t\r \u00a0replicate\t\r \u00a0in\t\r \u00a0mouse\t\r \u00a0but\t\r \u00a0not\t\r \u00a0human\t\r \u00a0macrophages(452).\t\r \u00a0It\t\r \u00a0would\t\r \u00a0be\t\r \u00a0interesting\t\r \u00a0to\t\r \u00a0induce\t\r \u00a0uptake\t\r \u00a0of\t\r \u00a0opsonized\t\r \u00a0Salmonella\t\r \u00a0by\t\r \u00a0these\t\r \u00a0cell\t\r \u00a0lines\t\r \u00a0and\t\r \u00a0compare\t\r \u00a0protein\t\r \u00a0 \t\r \u00a0\t\r \u00a0 141\t\r \u00a0 phosphorylation\t\r \u00a0dynamics\t\r \u00a0between\t\r \u00a0these\t\r \u00a0models\t\r \u00a0of\t\r \u00a0gastroenteritis\t\r \u00a0and\t\r \u00a0enteric\t\r \u00a0fever.\t\r \u00a0Studies\t\r \u00a0in\t\r \u00a0macrophages\t\r \u00a0would\t\r \u00a0likely\t\r \u00a0complicate\t\r \u00a0the\t\r \u00a0analysis\t\r \u00a0however,\t\r \u00a0as\t\r \u00a0several\t\r \u00a0innate\t\r \u00a0immune\t\r \u00a0receptors\t\r \u00a0are\t\r \u00a0activated\t\r \u00a0by\t\r \u00a0PAMPs\t\r \u00a0in\t\r \u00a0these\t\r \u00a0cells,\t\r \u00a0potentially\t\r \u00a0obscuring\t\r \u00a0signaling\t\r \u00a0downstream\t\r \u00a0of\t\r \u00a0host\t\r \u00a0receptors\t\r \u00a0versus\t\r \u00a0bacterial\t\r \u00a0effectors.\t\r \u00a0Furthermore,\t\r \u00a0several\t\r \u00a0interesting\t\r \u00a0hypotheses\t\r \u00a0can\t\r \u00a0also\t\r \u00a0be\t\r \u00a0derived\t\r \u00a0from\t\r \u00a0specific\t\r \u00a0dynamic\t\r \u00a0proteins\t\r \u00a0within\t\r \u00a0the\t\r \u00a0phosphoproteomics\t\r \u00a0data.\t\r \u00a0These\t\r \u00a0include\t\r \u00a0additional\t\r \u00a0studies\t\r \u00a0into\t\r \u00a0the\t\r \u00a0phosphorylation\t\r \u00a0sites\t\r \u00a0identified\t\r \u00a0within\t\r \u00a0the\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0translocon\t\r \u00a0SipB\t\r \u00a0to\t\r \u00a0determine\t\r \u00a0whether\t\r \u00a0these\t\r \u00a0modifications\t\r \u00a0are\t\r \u00a0induced\t\r \u00a0by\t\r \u00a0the\t\r \u00a0host\t\r \u00a0or\t\r \u00a0by\t\r \u00a0the\t\r \u00a0bacteria,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0their\t\r \u00a0possible\t\r \u00a0role\t\r \u00a0in\t\r \u00a0translocon\t\r \u00a0function.\t\r \u00a0To\t\r \u00a0determine\t\r \u00a0whether\t\r \u00a0a\t\r \u00a0bacterial\t\r \u00a0kinase\t\r \u00a0phosphorylates\t\r \u00a0SipB,\t\r \u00a0phosphoproteomics\t\r \u00a0could\t\r \u00a0be\t\r \u00a0employed\t\r \u00a0on\t\r \u00a0bacterial\t\r \u00a0lysates\t\r \u00a0during\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0inducing\t\r \u00a0conditions(381,\t\r \u00a0382).\t\r \u00a0The\t\r \u00a0kinase\t\r \u00a0prediction\t\r \u00a0algorithm\t\r \u00a0used\t\r \u00a0in\t\r \u00a0Chapter\t\r \u00a04\t\r \u00a0could\t\r \u00a0also\t\r \u00a0be\t\r \u00a0employed\t\r \u00a0to\t\r \u00a0predict\t\r \u00a0the\t\r \u00a0human\t\r \u00a0kinases\t\r \u00a0most\t\r \u00a0likely\t\r \u00a0to\t\r \u00a0target\t\r \u00a0these\t\r \u00a0sites.\t\r \u00a0To\t\r \u00a0determine\t\r \u00a0whether\t\r \u00a0these\t\r \u00a0sites\t\r \u00a0regulate\t\r \u00a0translocon\t\r \u00a0assembly\t\r \u00a0and\t\r \u00a0function,\t\r \u00a0an\t\r \u00a0S.\t\r \u00a0Typhimurium\t\r \u00a0strain\t\r \u00a0mutant\t\r \u00a0for\t\r \u00a0SipB\t\r \u00a0could\t\r \u00a0be\t\r \u00a0made\t\r \u00a0and\t\r \u00a0re-\u00ad\u2010complemented\t\r \u00a0with\t\r \u00a0WT\t\r \u00a0SipB,\t\r \u00a0as\t\r \u00a0well\t\r \u00a0as\t\r \u00a0constructs\t\r \u00a0where\t\r \u00a0these\t\r \u00a0sites\t\r \u00a0are\t\r \u00a0mutated\t\r \u00a0to\t\r \u00a0alanine\t\r \u00a0or\t\r \u00a0aspartic\t\r \u00a0acid.\t\r \u00a0Localization\t\r \u00a0of\t\r \u00a0SipB\t\r \u00a0within\t\r \u00a0bacteria\t\r \u00a0and\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0could\t\r \u00a0then\t\r \u00a0be\t\r \u00a0determined\t\r \u00a0using\t\r \u00a0SipB\t\r \u00a0antibodies,\t\r \u00a0and\t\r \u00a0protein\t\r \u00a0translocation\t\r \u00a0could\t\r \u00a0be\t\r \u00a0assayed\t\r \u00a0by\t\r \u00a0imaging\t\r \u00a0tagged\t\r \u00a0T3SS-\u00ad\u20101\t\r \u00a0effectors\t\r \u00a0within\t\r \u00a0host\t\r \u00a0cells,\t\r \u00a0or\t\r \u00a0by\t\r \u00a0assaying\t\r \u00a0invasion\t\r \u00a0efficiency(133).\t\r \u00a0Several\t\r \u00a0dynamic\t\r \u00a0phosphorylation\t\r \u00a0sites\t\r \u00a0identified\t\r \u00a0in\t\r \u00a0host\t\r \u00a0proteins\t\r \u00a0during\t\r \u00a0S.\t\r \u00a0Typhimurium\t\r \u00a0infection\t\r \u00a0could\t\r \u00a0also\t\r \u00a0initiate\t\r \u00a0further\t\r \u00a0research.\t\r \u00a0As\t\r \u00a0both\t\r \u00a0Akt\t\r \u00a0and\t\r \u00a0PKC\t\r \u00a0were\t\r \u00a0identified\t\r \u00a0as\t\r \u00a0global\t\r \u00a0regulators\t\r \u00a0during\t\r \u00a0the\t\r \u00a0initial\t\r \u00a0stages\t\r \u00a0of\t\r \u00a0infection\t\r \u00a0(Chapter\t\r \u00a04),\t\r \u00a0the\t\r \u00a0functions\t\r \u00a0of\t\r \u00a0several\t\r \u00a0sites\t\r \u00a0downstream\t\r \u00a0of\t\r \u00a0these\t\r \u00a0kinases\t\r \u00a0will\t\r \u00a0likely\t\r \u00a0reveal\t\r \u00a0novel\t\r \u00a0 \t\r \u00a0\t\r \u00a0 142\t\r \u00a0 host\t\r \u00a0processes\t\r \u00a0targeted\t\r \u00a0by\t\r \u00a0Salmonella.\t\r \u00a0Activating\t\r \u00a0sites\t\r \u00a0in\t\r \u00a0both\t\r \u00a0IQGAP1\t\r \u00a0and\t\r \u00a0PRKD3\t\r \u00a0were\t\r \u00a0identified,\t\r \u00a0both\t\r \u00a0of\t\r \u00a0which\t\r \u00a0are\t\r \u00a0known\t\r \u00a0targets\t\r \u00a0of\t\r \u00a0PKC\t\r \u00a0and\t\r \u00a0thus\t\r \u00a0likely\t\r \u00a0also\t\r \u00a0dependent\t\r \u00a0on\t\r \u00a0SipA(147,\t\r \u00a0516,\t\r \u00a0517).\t\r \u00a0However,\t\r \u00a0their\t\r \u00a0potential\t\r \u00a0roles\t\r \u00a0during\t\r \u00a0infection\t\r \u00a0are\t\r \u00a0completely\t\r \u00a0unknown.\t\r \u00a0Downstream\t\r \u00a0of\t\r \u00a0SopB,\t\r \u00a0an\t\r \u00a0activating\t\r \u00a0Akt\t\r \u00a0site\t\r \u00a0in\t\r \u00a0PFKFB2\t\r \u00a0was\t\r \u00a0also\t\r \u00a0identified.\t\r \u00a0PFKFB2\t\r \u00a0generates\t\r \u00a0fructose\t\r \u00a02,6-\u00ad\u2010bisphosphate\t\r \u00a0during\t\r \u00a0glycolysis,\t\r \u00a0suggesting\t\r \u00a0that,\t\r \u00a0through\t\r \u00a0Akt,\t\r \u00a0SopB\t\r \u00a0may\t\r \u00a0regulate\t\r \u00a0carbohydrate\t\r \u00a0metabolism(539).\t\r \u00a0However,\t\r \u00a0the\t\r \u00a0potential\t\r \u00a0role\t\r \u00a0that\t\r \u00a0this\t\r \u00a0enzyme\t\r \u00a0plays\t\r \u00a0in\t\r \u00a0establishing\t\r \u00a0Salmonella\u2019s\t\r \u00a0replicative\t\r \u00a0niche\t\r \u00a0needs\t\r \u00a0further\t\r \u00a0investigation.\t\r \u00a0\t\r \u00a0 Ultimately,\t\r \u00a0the\t\r \u00a0research\t\r \u00a0presented\t\r \u00a0here\t\r \u00a0has\t\r \u00a0furthered\t\r \u00a0our\t\r \u00a0understanding\t\r \u00a0of\t\r \u00a0how\t\r \u00a0the\t\r \u00a0intracellular\t\r \u00a0bacterial\t\r \u00a0pathogen\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0interacts\t\r \u00a0with\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0to\t\r \u00a0cause\t\r \u00a0disease.\t\r \u00a0This\t\r \u00a0work\t\r \u00a0has\t\r \u00a0identified\t\r \u00a0several\t\r \u00a0novel\t\r \u00a0host\t\r \u00a0targets\t\r \u00a0of\t\r \u00a0the\t\r \u00a0 Salmonella\t\r \u00a0effector\t\r \u00a0SopB,\t\r \u00a0while\t\r \u00a0also\t\r \u00a0providing\t\r \u00a0a\t\r \u00a0global\t\r \u00a0view\t\r \u00a0of\t\r \u00a0host\t\r \u00a0phosphorylation\t\r \u00a0signaling\t\r \u00a0during\t\r \u00a0bacterial\t\r \u00a0infection,\t\r \u00a0and\t\r \u00a0developing\t\r \u00a0proteomics\t\r \u00a0techniques\t\r \u00a0which\t\r \u00a0are\t\r \u00a0widely\t\r \u00a0applicable\t\r \u00a0to\t\r \u00a0future\t\r \u00a0research\t\r \u00a0within\t\r \u00a0the\t\r \u00a0field\t\r \u00a0of\t\r \u00a0pathogen-\u00ad\u2010host\t\r \u00a0interactions.\t\r \u00a0The\t\r \u00a0data\t\r \u00a0and\t\r \u00a0methodologies\t\r \u00a0presented\t\r \u00a0here\t\r \u00a0have\t\r \u00a0contributed\t\r \u00a0significantly\t\r \u00a0to\t\r \u00a0these\t\r \u00a0fields\t\r \u00a0and,\t\r \u00a0perhaps\t\r \u00a0with\t\r \u00a0a\t\r \u00a0few\t\r \u00a0updates\t\r \u00a0and\t\r \u00a0improvements,\t\r \u00a0lay\t\r \u00a0the\t\r \u00a0groundwork\t\r \u00a0for\t\r \u00a0several\t\r \u00a0new\t\r \u00a0and\t\r \u00a0exciting\t\r \u00a0avenues\t\r \u00a0of\t\r \u00a0research.\t\r \u00a0 \t\r \u00a0\t\r \u00a0 143\t\r \u00a0 References\t\r \u00a0 1.\t\r \u00a0 P.\t\r \u00a0R.\t\r \u00a0Murray,\t\r \u00a0K.\t\r \u00a0S.\t\r \u00a0Rosenthal,\t\r \u00a0M.\t\r \u00a0A.\t\r \u00a0Pfaller,\t\r \u00a0Medical\t\r \u00a0Microbiology,\t\r \u00a06th\t\r \u00a0Edition.\t\r \u00a0\t\r \u00a0(Mosby\t\r \u00a0Elsevier,\t\r \u00a0Philadelphia,\t\r \u00a02009).\t\r \u00a02.\t\r \u00a0 M.\t\r \u00a0Kennedy,\t\r \u00a0R.\t\r \u00a0Villar,\t\r \u00a0D.\t\r \u00a0J.\t\r \u00a0Vugia,\t\r \u00a0T.\t\r \u00a0Rabatsky-\u00ad\u2010Ehr,\t\r \u00a0M.\t\r \u00a0M.\t\r \u00a0Farley,\t\r \u00a0M.\t\r \u00a0Pass,\t\r \u00a0K.\t\r \u00a0Smith,\t\r \u00a0P.\t\r \u00a0Smith,\t\r \u00a0P.\t\r \u00a0R.\t\r \u00a0Cieslak,\t\r \u00a0B.\t\r \u00a0Imhoff,\t\r \u00a0P.\t\r \u00a0M.\t\r \u00a0Griffin,\t\r \u00a0Hospitalizations\t\r \u00a0and\t\r \u00a0deaths\t\r \u00a0due\t\r \u00a0to\t\r \u00a0Salmonella\t\r \u00a0infections,\t\r \u00a0FoodNet,\t\r \u00a01996-\u00ad\u20101999.\t\r \u00a0Clin\t\r \u00a0Infect\t\r \u00a0Dis\t\r \u00a038\t\r \u00a0 Suppl\t\r \u00a03,\t\r \u00a0S142-\u00ad\u2010148\t\r \u00a0(2004).\t\r \u00a03.\t\r \u00a0 J.\t\r \u00a0A.\t\r \u00a0Crump,\t\r \u00a0S.\t\r \u00a0P.\t\r \u00a0Luby,\t\r \u00a0E.\t\r \u00a0D.\t\r \u00a0Mintz,\t\r \u00a0The\t\r \u00a0global\t\r \u00a0burden\t\r \u00a0of\t\r \u00a0typhoid\t\r \u00a0fever.\t\r \u00a0Bull\t\r \u00a0 World\t\r \u00a0Health\t\r \u00a0Organ\t\r \u00a082,\t\r \u00a0346-\u00ad\u2010353\t\r \u00a0(2004).\t\r \u00a04.\t\r \u00a0 D.\t\r \u00a0DeRoeck,\t\r \u00a0L.\t\r \u00a0Jodar,\t\r \u00a0J.\t\r \u00a0Clemens,\t\r \u00a0Putting\t\r \u00a0typhoid\t\r \u00a0vaccination\t\r \u00a0on\t\r \u00a0the\t\r \u00a0global\t\r \u00a0health\t\r \u00a0agenda.\t\r \u00a0N\t\r \u00a0Engl\t\r \u00a0J\t\r \u00a0Med\t\r \u00a0357,\t\r \u00a01069-\u00ad\u20101071\t\r \u00a0(2007).\t\r \u00a05.\t\r \u00a0 C.\t\r \u00a0M.\t\r \u00a0Parry,\t\r \u00a0E.\t\r \u00a0J.\t\r \u00a0Threlfall,\t\r \u00a0Antimicrobial\t\r \u00a0resistance\t\r \u00a0in\t\r \u00a0typhoidal\t\r \u00a0and\t\r \u00a0nontyphoidal\t\r \u00a0salmonellae.\t\r \u00a0Curr\t\r \u00a0Opin\t\r \u00a0Infect\t\r \u00a0Dis\t\r \u00a021,\t\r \u00a0531-\u00ad\u2010538\t\r \u00a0(2008).\t\r \u00a06.\t\r \u00a0 S.\t\r \u00a0Joshi,\t\r \u00a0S.\t\r \u00a0K.\t\r \u00a0Amarnath,\t\r \u00a0Fluoroquinolone\t\r \u00a0resistance\t\r \u00a0in\t\r \u00a0Salmonella\t\r \u00a0typhi\t\r \u00a0and\t\r \u00a0S.\t\r \u00a0paratyphi\t\r \u00a0A\t\r \u00a0in\t\r \u00a0Bangalore,\t\r \u00a0India.\t\r \u00a0Trans\t\r \u00a0R\t\r \u00a0Soc\t\r \u00a0Trop\t\r \u00a0Med\t\r \u00a0Hyg\t\r \u00a0101,\t\r \u00a0308-\u00ad\u2010310\t\r \u00a0(2007).\t\r \u00a07.\t\r \u00a0 K.\t\r \u00a0O.\t\r \u00a0Akinyemi,\t\r \u00a0A.\t\r \u00a0O.\t\r \u00a0Coker,\t\r \u00a0Trends\t\r \u00a0of\t\r \u00a0antibiotic\t\r \u00a0resistance\t\r \u00a0in\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0typhi\t\r \u00a0isolated\t\r \u00a0from\t\r \u00a0hospitalized\t\r \u00a0patients\t\r \u00a0from\t\r \u00a01997\t\r \u00a0to\t\r \u00a02004\t\r \u00a0in\t\r \u00a0Lagos,\t\r \u00a0Nigeria.\t\r \u00a0Indian\t\r \u00a0J\t\r \u00a0Med\t\r \u00a0Microbiol\t\r \u00a025,\t\r \u00a0436-\u00ad\u2010437\t\r \u00a0(2007).\t\r \u00a08.\t\r \u00a0 A.\t\r \u00a0P.\t\r \u00a0Maskey,\t\r \u00a0B.\t\r \u00a0Basnyat,\t\r \u00a0G.\t\r \u00a0E.\t\r \u00a0Thwaites,\t\r \u00a0J.\t\r \u00a0I.\t\r \u00a0Campbell,\t\r \u00a0J.\t\r \u00a0J.\t\r \u00a0Farrar,\t\r \u00a0M.\t\r \u00a0D.\t\r \u00a0Zimmerman,\t\r \u00a0Emerging\t\r \u00a0trends\t\r \u00a0in\t\r \u00a0enteric\t\r \u00a0fever\t\r \u00a0in\t\r \u00a0Nepal:\t\r \u00a09124\t\r \u00a0cases\t\r \u00a0confirmed\t\r \u00a0by\t\r \u00a0blood\t\r \u00a0culture\t\r \u00a01993-\u00ad\u20102003.\t\r \u00a0Trans\t\r \u00a0R\t\r \u00a0Soc\t\r \u00a0Trop\t\r \u00a0Med\t\r \u00a0Hyg\t\r \u00a0102,\t\r \u00a091-\u00ad\u201095\t\r \u00a0(2008).\t\r \u00a09.\t\r \u00a0 J.\t\r \u00a0W.\t\r \u00a0Foster,\t\r \u00a0H.\t\r \u00a0K.\t\r \u00a0Hall,\t\r \u00a0Adaptive\t\r \u00a0acidification\t\r \u00a0tolerance\t\r \u00a0response\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0typhimurium.\t\r \u00a0J\t\r \u00a0Bacteriol\t\r \u00a0172,\t\r \u00a0771-\u00ad\u2010778\t\r \u00a0(1990).\t\r \u00a010.\t\r \u00a0 F.\t\r \u00a0Garcia-\u00ad\u2010del\t\r \u00a0Portillo,\t\r \u00a0J.\t\r \u00a0W.\t\r \u00a0Foster,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0Role\t\r \u00a0of\t\r \u00a0acid\t\r \u00a0tolerance\t\r \u00a0response\t\r \u00a0genes\t\r \u00a0in\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0virulence.\t\r \u00a0Infect\t\r \u00a0Immun\t\r \u00a061,\t\r \u00a04489-\u00ad\u20104492\t\r \u00a0(1993).\t\r \u00a011.\t\r \u00a0 A.\t\r \u00a0M.\t\r \u00a0Prouty,\t\r \u00a0I.\t\r \u00a0E.\t\r \u00a0Brodsky,\t\r \u00a0S.\t\r \u00a0Falkow,\t\r \u00a0J.\t\r \u00a0S.\t\r \u00a0Gunn,\t\r \u00a0Bile-\u00ad\u2010salt-\u00ad\u2010mediated\t\r \u00a0induction\t\r \u00a0of\t\r \u00a0antimicrobial\t\r \u00a0and\t\r \u00a0bile\t\r \u00a0resistance\t\r \u00a0in\t\r \u00a0Salmonella\t\r \u00a0typhimurium.\t\r \u00a0Microbiology\t\r \u00a0 150,\t\r \u00a0775-\u00ad\u2010783\t\r \u00a0(2004).\t\r \u00a012.\t\r \u00a0 P.\t\r \u00a0Michetti,\t\r \u00a0M.\t\r \u00a0J.\t\r \u00a0Mahan,\t\r \u00a0J.\t\r \u00a0M.\t\r \u00a0Slauch,\t\r \u00a0J.\t\r \u00a0J.\t\r \u00a0Mekalanos,\t\r \u00a0M.\t\r \u00a0R.\t\r \u00a0Neutra,\t\r \u00a0Monoclonal\t\r \u00a0secretory\t\r \u00a0immunoglobulin\t\r \u00a0A\t\r \u00a0protects\t\r \u00a0mice\t\r \u00a0against\t\r \u00a0oral\t\r \u00a0challenge\t\r \u00a0with\t\r \u00a0the\t\r \u00a0invasive\t\r \u00a0pathogen\t\r \u00a0Salmonella\t\r \u00a0typhimurium.\t\r \u00a0Infect\t\r \u00a0Immun\t\r \u00a060,\t\r \u00a01786-\u00ad\u20101792\t\r \u00a0(1992).\t\r \u00a013.\t\r \u00a0 M.\t\r \u00a0E.\t\r \u00a0Selsted,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0A.\t\r \u00a0H.\t\r \u00a0Henschen,\t\r \u00a0A.\t\r \u00a0J.\t\r \u00a0Ouellette,\t\r \u00a0Enteric\t\r \u00a0defensins:\t\r \u00a0antibiotic\t\r \u00a0peptide\t\r \u00a0components\t\r \u00a0of\t\r \u00a0intestinal\t\r \u00a0host\t\r \u00a0defense.\t\r \u00a0J\t\r \u00a0Cell\t\r \u00a0Biol\t\r \u00a0118,\t\r \u00a0929-\u00ad\u2010936\t\r \u00a0(1992).\t\r \u00a014.\t\r \u00a0 A.\t\r \u00a0W.\t\r \u00a0van\t\r \u00a0der\t\r \u00a0Velden,\t\r \u00a0A.\t\r \u00a0J.\t\r \u00a0Baumler,\t\r \u00a0R.\t\r \u00a0M.\t\r \u00a0Tsolis,\t\r \u00a0F.\t\r \u00a0Heffron,\t\r \u00a0Multiple\t\r \u00a0fimbrial\t\r \u00a0adhesins\t\r \u00a0are\t\r \u00a0required\t\r \u00a0for\t\r \u00a0full\t\r \u00a0virulence\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0in\t\r \u00a0mice.\t\r \u00a0 Infect\t\r \u00a0Immun\t\r \u00a066,\t\r \u00a02803-\u00ad\u20102808\t\r \u00a0(1998).\t\r \u00a015.\t\r \u00a0 A.\t\r \u00a0J.\t\r \u00a0Baumler,\t\r \u00a0R.\t\r \u00a0M.\t\r \u00a0Tsolis,\t\r \u00a0F.\t\r \u00a0Heffron,\t\r \u00a0Contribution\t\r \u00a0of\t\r \u00a0fimbrial\t\r \u00a0operons\t\r \u00a0to\t\r \u00a0attachment\t\r \u00a0to\t\r \u00a0and\t\r \u00a0invasion\t\r \u00a0of\t\r \u00a0epithelial\t\r \u00a0cell\t\r \u00a0lines\t\r \u00a0by\t\r \u00a0Salmonella\t\r \u00a0typhimurium.\t\r \u00a0Infect\t\r \u00a0Immun\t\r \u00a064,\t\r \u00a01862-\u00ad\u20101865\t\r \u00a0(1996).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 144\t\r \u00a0 16.\t\r \u00a0 B.\t\r \u00a0D.\t\r \u00a0Jones,\t\r \u00a0N.\t\r \u00a0Ghori,\t\r \u00a0S.\t\r \u00a0Falkow,\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0initiates\t\r \u00a0murine\t\r \u00a0infection\t\r \u00a0by\t\r \u00a0penetrating\t\r \u00a0and\t\r \u00a0destroying\t\r \u00a0the\t\r \u00a0specialized\t\r \u00a0epithelial\t\r \u00a0M\t\r \u00a0cells\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Peyer's\t\r \u00a0patches.\t\r \u00a0J\t\r \u00a0Exp\t\r \u00a0Med\t\r \u00a0180,\t\r \u00a015-\u00ad\u201023\t\r \u00a0(1994).\t\r \u00a017.\t\r \u00a0 A.\t\r \u00a0Takeuchi,\t\r \u00a0Electron\t\r \u00a0microscope\t\r \u00a0studies\t\r \u00a0of\t\r \u00a0experimental\t\r \u00a0Salmonella\t\r \u00a0infection.\t\r \u00a0I.\t\r \u00a0Penetration\t\r \u00a0into\t\r \u00a0the\t\r \u00a0intestinal\t\r \u00a0epithelium\t\r \u00a0by\t\r \u00a0Salmonella\t\r \u00a0typhimurium.\t\r \u00a0Am\t\r \u00a0J\t\r \u00a0Pathol\t\r \u00a050,\t\r \u00a0109-\u00ad\u2010136\t\r \u00a0(1967).\t\r \u00a018.\t\r \u00a0 A.\t\r \u00a0Vazquez-\u00ad\u2010Torres,\t\r \u00a0J.\t\r \u00a0Jones-\u00ad\u2010Carson,\t\r \u00a0A.\t\r \u00a0J.\t\r \u00a0Baumler,\t\r \u00a0S.\t\r \u00a0Falkow,\t\r \u00a0R.\t\r \u00a0Valdivia,\t\r \u00a0W.\t\r \u00a0Brown,\t\r \u00a0M.\t\r \u00a0Le,\t\r \u00a0R.\t\r \u00a0Berggren,\t\r \u00a0W.\t\r \u00a0T.\t\r \u00a0Parks,\t\r \u00a0F.\t\r \u00a0C.\t\r \u00a0Fang,\t\r \u00a0Extraintestinal\t\r \u00a0dissemination\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0by\t\r \u00a0CD18-\u00ad\u2010expressing\t\r \u00a0phagocytes.\t\r \u00a0Nature\t\r \u00a0401,\t\r \u00a0804-\u00ad\u2010808\t\r \u00a0(1999).\t\r \u00a019.\t\r \u00a0 M.\t\r \u00a0Rescigno,\t\r \u00a0M.\t\r \u00a0Urbano,\t\r \u00a0B.\t\r \u00a0Valzasina,\t\r \u00a0M.\t\r \u00a0Francolini,\t\r \u00a0G.\t\r \u00a0Rotta,\t\r \u00a0R.\t\r \u00a0Bonasio,\t\r \u00a0F.\t\r \u00a0Granucci,\t\r \u00a0J.\t\r \u00a0P.\t\r \u00a0Kraehenbuhl,\t\r \u00a0P.\t\r \u00a0Ricciardi-\u00ad\u2010Castagnoli,\t\r \u00a0Dendritic\t\r \u00a0cells\t\r \u00a0express\t\r \u00a0tight\t\r \u00a0junction\t\r \u00a0proteins\t\r \u00a0and\t\r \u00a0penetrate\t\r \u00a0gut\t\r \u00a0epithelial\t\r \u00a0monolayers\t\r \u00a0to\t\r \u00a0sample\t\r \u00a0bacteria.\t\r \u00a0Nat\t\r \u00a0Immunol\t\r \u00a02,\t\r \u00a0361-\u00ad\u2010367\t\r \u00a0(2001).\t\r \u00a020.\t\r \u00a0 M.\t\r \u00a0A.\t\r \u00a0Jepson,\t\r \u00a0C.\t\r \u00a0B.\t\r \u00a0Collares-\u00ad\u2010Buzato,\t\r \u00a0M.\t\r \u00a0A.\t\r \u00a0Clark,\t\r \u00a0B.\t\r \u00a0H.\t\r \u00a0Hirst,\t\r \u00a0N.\t\r \u00a0L.\t\r \u00a0Simmons,\t\r \u00a0Rapid\t\r \u00a0disruption\t\r \u00a0of\t\r \u00a0epithelial\t\r \u00a0barrier\t\r \u00a0function\t\r \u00a0by\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0is\t\r \u00a0associated\t\r \u00a0with\t\r \u00a0structural\t\r \u00a0modification\t\r \u00a0of\t\r \u00a0intercellular\t\r \u00a0junctions.\t\r \u00a0Infect\t\r \u00a0 Immun\t\r \u00a063,\t\r \u00a0356-\u00ad\u2010359\t\r \u00a0(1995).\t\r \u00a021.\t\r \u00a0 A.\t\r \u00a0Haraga,\t\r \u00a0M.\t\r \u00a0B.\t\r \u00a0Ohlson,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0Salmonellae\t\r \u00a0interplay\t\r \u00a0with\t\r \u00a0host\t\r \u00a0cells.\t\r \u00a0Nat\t\r \u00a0 Rev\t\r \u00a0Microbiol\t\r \u00a06,\t\r \u00a053-\u00ad\u201066\t\r \u00a0(2008).\t\r \u00a022.\t\r \u00a0 B.\t\r \u00a0A.\t\r \u00a0McCormick,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0D.\t\r \u00a0Carnes,\t\r \u00a0J.\t\r \u00a0L.\t\r \u00a0Madara,\t\r \u00a0Transepithelial\t\r \u00a0signaling\t\r \u00a0to\t\r \u00a0neutrophils\t\r \u00a0by\t\r \u00a0salmonellae:\t\r \u00a0a\t\r \u00a0novel\t\r \u00a0virulence\t\r \u00a0mechanism\t\r \u00a0for\t\r \u00a0gastroenteritis.\t\r \u00a0Infect\t\r \u00a0Immun\t\r \u00a063,\t\r \u00a02302-\u00ad\u20102309\t\r \u00a0(1995).\t\r \u00a023.\t\r \u00a0 J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0R.\t\r \u00a0Curtiss,\t\r \u00a03rd,\t\r \u00a0Cloning\t\r \u00a0and\t\r \u00a0molecular\t\r \u00a0characterization\t\r \u00a0of\t\r \u00a0genes\t\r \u00a0whose\t\r \u00a0products\t\r \u00a0allow\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0to\t\r \u00a0penetrate\t\r \u00a0tissue\t\r \u00a0culture\t\r \u00a0cells.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a086,\t\r \u00a06383-\u00ad\u20106387\t\r \u00a0(1989).\t\r \u00a024.\t\r \u00a0 B.\t\r \u00a0D.\t\r \u00a0Jones,\t\r \u00a0S.\t\r \u00a0Falkow,\t\r \u00a0Identification\t\r \u00a0and\t\r \u00a0characterization\t\r \u00a0of\t\r \u00a0a\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0oxygen-\u00ad\u2010regulated\t\r \u00a0gene\t\r \u00a0required\t\r \u00a0for\t\r \u00a0bacterial\t\r \u00a0internalization.\t\r \u00a0 Infect\t\r \u00a0Immun\t\r \u00a062,\t\r \u00a03745-\u00ad\u20103752\t\r \u00a0(1994).\t\r \u00a025.\t\r \u00a0 A.\t\r \u00a0J.\t\r \u00a0Baumler,\t\r \u00a0R.\t\r \u00a0M.\t\r \u00a0Tsolis,\t\r \u00a0P.\t\r \u00a0J.\t\r \u00a0Valentine,\t\r \u00a0T.\t\r \u00a0A.\t\r \u00a0Ficht,\t\r \u00a0F.\t\r \u00a0Heffron,\t\r \u00a0Synergistic\t\r \u00a0effect\t\r \u00a0of\t\r \u00a0mutations\t\r \u00a0in\t\r \u00a0invA\t\r \u00a0and\t\r \u00a0lpfC\t\r \u00a0on\t\r \u00a0the\t\r \u00a0ability\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0to\t\r \u00a0cause\t\r \u00a0murine\t\r \u00a0typhoid.\t\r \u00a0Infect\t\r \u00a0Immun\t\r \u00a065,\t\r \u00a02254-\u00ad\u20102259\t\r \u00a0(1997).\t\r \u00a026.\t\r \u00a0 S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0A.\t\r \u00a0M.\t\r \u00a0Kukral,\t\r \u00a0J.\t\r \u00a0J.\t\r \u00a0Mekalanos,\t\r \u00a0A\t\r \u00a0two-\u00ad\u2010component\t\r \u00a0regulatory\t\r \u00a0system\t\r \u00a0(phoP\t\r \u00a0phoQ)\t\r \u00a0controls\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0virulence.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0 U\t\r \u00a0S\t\r \u00a0A\t\r \u00a086,\t\r \u00a05054-\u00ad\u20105058\t\r \u00a0(1989).\t\r \u00a027.\t\r \u00a0 C.\t\r \u00a0M.\t\r \u00a0Alpuche\t\r \u00a0Aranda,\t\r \u00a0J.\t\r \u00a0A.\t\r \u00a0Swanson,\t\r \u00a0W.\t\r \u00a0P.\t\r \u00a0Loomis,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0activates\t\r \u00a0virulence\t\r \u00a0gene\t\r \u00a0transcription\t\r \u00a0within\t\r \u00a0acidified\t\r \u00a0macrophage\t\r \u00a0phagosomes.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a089,\t\r \u00a010079-\u00ad\u201010083\t\r \u00a0(1992).\t\r \u00a028.\t\r \u00a0 C.\t\r \u00a0M.\t\r \u00a0Alpuche-\u00ad\u2010Aranda,\t\r \u00a0E.\t\r \u00a0L.\t\r \u00a0Racoosin,\t\r \u00a0J.\t\r \u00a0A.\t\r \u00a0Swanson,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0Salmonella\t\r \u00a0stimulate\t\r \u00a0macrophage\t\r \u00a0macropinocytosis\t\r \u00a0and\t\r \u00a0persist\t\r \u00a0within\t\r \u00a0spacious\t\r \u00a0phagosomes.\t\r \u00a0J\t\r \u00a0Exp\t\r \u00a0Med\t\r \u00a0179,\t\r \u00a0601-\u00ad\u2010608\t\r \u00a0(1994).\t\r \u00a029.\t\r \u00a0 H.\t\r \u00a0Ochman,\t\r \u00a0F.\t\r \u00a0C.\t\r \u00a0Soncini,\t\r \u00a0F.\t\r \u00a0Solomon,\t\r \u00a0E.\t\r \u00a0A.\t\r \u00a0Groisman,\t\r \u00a0Identification\t\r \u00a0of\t\r \u00a0a\t\r \u00a0pathogenicity\t\r \u00a0island\t\r \u00a0required\t\r \u00a0for\t\r \u00a0Salmonella\t\r \u00a0survival\t\r \u00a0in\t\r \u00a0host\t\r \u00a0cells.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0 Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a093,\t\r \u00a07800-\u00ad\u20107804\t\r \u00a0(1996).\t\r \u00a030.\t\r \u00a0 C.\t\r \u00a0M.\t\r \u00a0Parry,\t\r \u00a0T.\t\r \u00a0T.\t\r \u00a0Hien,\t\r \u00a0G.\t\r \u00a0Dougan,\t\r \u00a0N.\t\r \u00a0J.\t\r \u00a0White,\t\r \u00a0J.\t\r \u00a0J.\t\r \u00a0Farrar,\t\r \u00a0Typhoid\t\r \u00a0fever.\t\r \u00a0N\t\r \u00a0Engl\t\r \u00a0 J\t\r \u00a0Med\t\r \u00a0347,\t\r \u00a01770-\u00ad\u20101782\t\r \u00a0(2002).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 145\t\r \u00a0 31.\t\r \u00a0 J.\t\r \u00a0Pace,\t\r \u00a0M.\t\r \u00a0J.\t\r \u00a0Hayman,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Signal\t\r \u00a0transduction\t\r \u00a0and\t\r \u00a0invasion\t\r \u00a0of\t\r \u00a0epithelial\t\r \u00a0cells\t\r \u00a0by\t\r \u00a0S.\t\r \u00a0typhimurium.\t\r \u00a0Cell\t\r \u00a072,\t\r \u00a0505-\u00ad\u2010514\t\r \u00a0(1993).\t\r \u00a032.\t\r \u00a0 I.\t\r \u00a0Rosenshine,\t\r \u00a0S.\t\r \u00a0Ruschkowski,\t\r \u00a0V.\t\r \u00a0Foubister,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0invasion\t\r \u00a0of\t\r \u00a0epithelial\t\r \u00a0cells:\t\r \u00a0role\t\r \u00a0of\t\r \u00a0induced\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0tyrosine\t\r \u00a0protein\t\r \u00a0phosphorylation.\t\r \u00a0Infect\t\r \u00a0Immun\t\r \u00a062,\t\r \u00a04969-\u00ad\u20104974\t\r \u00a0(1994).\t\r \u00a033.\t\r \u00a0 S.\t\r \u00a0Hobbie,\t\r \u00a0L.\t\r \u00a0M.\t\r \u00a0Chen,\t\r \u00a0R.\t\r \u00a0J.\t\r \u00a0Davis,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Involvement\t\r \u00a0of\t\r \u00a0mitogen-\u00ad\u2010activated\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a0pathways\t\r \u00a0in\t\r \u00a0the\t\r \u00a0nuclear\t\r \u00a0responses\t\r \u00a0and\t\r \u00a0cytokine\t\r \u00a0production\t\r \u00a0induced\t\r \u00a0by\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0in\t\r \u00a0cultured\t\r \u00a0intestinal\t\r \u00a0epithelial\t\r \u00a0cells.\t\r \u00a0J\t\r \u00a0 Immunol\t\r \u00a0159,\t\r \u00a05550-\u00ad\u20105559\t\r \u00a0(1997).\t\r \u00a034.\t\r \u00a0 J.\t\r \u00a0M.\t\r \u00a0Kim,\t\r \u00a0L.\t\r \u00a0Eckmann,\t\r \u00a0T.\t\r \u00a0C.\t\r \u00a0Savidge,\t\r \u00a0D.\t\r \u00a0C.\t\r \u00a0Lowe,\t\r \u00a0T.\t\r \u00a0Witthoft,\t\r \u00a0M.\t\r \u00a0F.\t\r \u00a0Kagnoff,\t\r \u00a0Apoptosis\t\r \u00a0of\t\r \u00a0human\t\r \u00a0intestinal\t\r \u00a0epithelial\t\r \u00a0cells\t\r \u00a0after\t\r \u00a0bacterial\t\r \u00a0invasion.\t\r \u00a0J\t\r \u00a0Clin\t\r \u00a0 Invest\t\r \u00a0102,\t\r \u00a01815-\u00ad\u20101823\t\r \u00a0(1998).\t\r \u00a035.\t\r \u00a0 G.\t\r \u00a0Paesold,\t\r \u00a0D.\t\r \u00a0G.\t\r \u00a0Guiney,\t\r \u00a0L.\t\r \u00a0Eckmann,\t\r \u00a0M.\t\r \u00a0F.\t\r \u00a0Kagnoff,\t\r \u00a0Genes\t\r \u00a0in\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0pathogenicity\t\r \u00a0island\t\r \u00a02\t\r \u00a0and\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0virulence\t\r \u00a0plasmid\t\r \u00a0are\t\r \u00a0essential\t\r \u00a0for\t\r \u00a0Salmonella-\u00ad\u2010induced\t\r \u00a0apoptosis\t\r \u00a0in\t\r \u00a0intestinal\t\r \u00a0epithelial\t\r \u00a0cells.\t\r \u00a0Cell\t\r \u00a0Microbiol\t\r \u00a04,\t\r \u00a0771-\u00ad\u2010781\t\r \u00a0(2002).\t\r \u00a036.\t\r \u00a0 H.\t\r \u00a0Zeng,\t\r \u00a0H.\t\r \u00a0Wu,\t\r \u00a0V.\t\r \u00a0Sloane,\t\r \u00a0R.\t\r \u00a0Jones,\t\r \u00a0Y.\t\r \u00a0Yu,\t\r \u00a0P.\t\r \u00a0Lin,\t\r \u00a0A.\t\r \u00a0T.\t\r \u00a0Gewirtz,\t\r \u00a0A.\t\r \u00a0S.\t\r \u00a0Neish,\t\r \u00a0Flagellin\/TLR5\t\r \u00a0responses\t\r \u00a0in\t\r \u00a0epithelia\t\r \u00a0reveal\t\r \u00a0intertwined\t\r \u00a0activation\t\r \u00a0of\t\r \u00a0inflammatory\t\r \u00a0and\t\r \u00a0apoptotic\t\r \u00a0pathways.\t\r \u00a0Am\t\r \u00a0J\t\r \u00a0Physiol\t\r \u00a0Gastrointest\t\r \u00a0Liver\t\r \u00a0Physiol\t\r \u00a0 290,\t\r \u00a0G96-\u00ad\u2010G108\t\r \u00a0(2006).\t\r \u00a037.\t\r \u00a0 S.\t\r \u00a0Mariathasan,\t\r \u00a0K.\t\r \u00a0Newton,\t\r \u00a0D.\t\r \u00a0M.\t\r \u00a0Monack,\t\r \u00a0D.\t\r \u00a0Vucic,\t\r \u00a0D.\t\r \u00a0M.\t\r \u00a0French,\t\r \u00a0W.\t\r \u00a0P.\t\r \u00a0Lee,\t\r \u00a0M.\t\r \u00a0Roose-\u00ad\u2010Girma,\t\r \u00a0S.\t\r \u00a0Erickson,\t\r \u00a0V.\t\r \u00a0M.\t\r \u00a0Dixit,\t\r \u00a0Differential\t\r \u00a0activation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0inflammasome\t\r \u00a0by\t\r \u00a0caspase-\u00ad\u20101\t\r \u00a0adaptors\t\r \u00a0ASC\t\r \u00a0and\t\r \u00a0Ipaf.\t\r \u00a0Nature\t\r \u00a0430,\t\r \u00a0213-\u00ad\u2010218\t\r \u00a0(2004).\t\r \u00a038.\t\r \u00a0 E.\t\r \u00a0A.\t\r \u00a0Miao,\t\r \u00a0C.\t\r \u00a0M.\t\r \u00a0Alpuche-\u00ad\u2010Aranda,\t\r \u00a0M.\t\r \u00a0Dors,\t\r \u00a0A.\t\r \u00a0E.\t\r \u00a0Clark,\t\r \u00a0M.\t\r \u00a0W.\t\r \u00a0Bader,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0A.\t\r \u00a0Aderem,\t\r \u00a0Cytoplasmic\t\r \u00a0flagellin\t\r \u00a0activates\t\r \u00a0caspase-\u00ad\u20101\t\r \u00a0and\t\r \u00a0secretion\t\r \u00a0of\t\r \u00a0interleukin\t\r \u00a01beta\t\r \u00a0via\t\r \u00a0Ipaf.\t\r \u00a0Nat\t\r \u00a0Immunol\t\r \u00a07,\t\r \u00a0569-\u00ad\u2010575\t\r \u00a0(2006).\t\r \u00a039.\t\r \u00a0 A.\t\r \u00a0W.\t\r \u00a0van\t\r \u00a0der\t\r \u00a0Velden,\t\r \u00a0M.\t\r \u00a0Velasquez,\t\r \u00a0M.\t\r \u00a0N.\t\r \u00a0Starnbach,\t\r \u00a0Salmonella\t\r \u00a0rapidly\t\r \u00a0kill\t\r \u00a0dendritic\t\r \u00a0cells\t\r \u00a0via\t\r \u00a0a\t\r \u00a0caspase-\u00ad\u20101-\u00ad\u2010dependent\t\r \u00a0mechanism.\t\r \u00a0J\t\r \u00a0Immunol\t\r \u00a0171,\t\r \u00a06742-\u00ad\u20106749\t\r \u00a0(2003).\t\r \u00a040.\t\r \u00a0 M.\t\r \u00a0A.\t\r \u00a0Brennan,\t\r \u00a0B.\t\r \u00a0T.\t\r \u00a0Cookson,\t\r \u00a0Salmonella\t\r \u00a0induces\t\r \u00a0macrophage\t\r \u00a0death\t\r \u00a0by\t\r \u00a0caspase-\u00ad\u20101-\u00ad\u2010dependent\t\r \u00a0necrosis.\t\r \u00a0Mol\t\r \u00a0Microbiol\t\r \u00a038,\t\r \u00a031-\u00ad\u201040\t\r \u00a0(2000).\t\r \u00a041.\t\r \u00a0 S.\t\r \u00a0L.\t\r \u00a0Fink,\t\r \u00a0B.\t\r \u00a0T.\t\r \u00a0Cookson,\t\r \u00a0Apoptosis,\t\r \u00a0pyroptosis,\t\r \u00a0and\t\r \u00a0necrosis:\t\r \u00a0mechanistic\t\r \u00a0description\t\r \u00a0of\t\r \u00a0dead\t\r \u00a0and\t\r \u00a0dying\t\r \u00a0eukaryotic\t\r \u00a0cells.\t\r \u00a0Infect\t\r \u00a0Immun\t\r \u00a073,\t\r \u00a01907-\u00ad\u20101916\t\r \u00a0(2005).\t\r \u00a042.\t\r \u00a0 S.\t\r \u00a0L.\t\r \u00a0Fink,\t\r \u00a0B.\t\r \u00a0T.\t\r \u00a0Cookson,\t\r \u00a0Caspase-\u00ad\u20101-\u00ad\u2010dependent\t\r \u00a0pore\t\r \u00a0formation\t\r \u00a0during\t\r \u00a0pyroptosis\t\r \u00a0leads\t\r \u00a0to\t\r \u00a0osmotic\t\r \u00a0lysis\t\r \u00a0of\t\r \u00a0infected\t\r \u00a0host\t\r \u00a0macrophages.\t\r \u00a0Cell\t\r \u00a0Microbiol\t\r \u00a0 8,\t\r \u00a01812-\u00ad\u20101825\t\r \u00a0(2006).\t\r \u00a043.\t\r \u00a0 M.\t\r \u00a0J.\t\r \u00a0Garner,\t\r \u00a0R.\t\r \u00a0D.\t\r \u00a0Hayward,\t\r \u00a0V.\t\r \u00a0Koronakis,\t\r \u00a0The\t\r \u00a0Salmonella\t\r \u00a0pathogenicity\t\r \u00a0island\t\r \u00a01\t\r \u00a0secretion\t\r \u00a0system\t\r \u00a0directs\t\r \u00a0cellular\t\r \u00a0cholesterol\t\r \u00a0redistribution\t\r \u00a0during\t\r \u00a0mammalian\t\r \u00a0cell\t\r \u00a0entry\t\r \u00a0and\t\r \u00a0intracellular\t\r \u00a0trafficking.\t\r \u00a0Cell\t\r \u00a0Microbiol\t\r \u00a04,\t\r \u00a0153-\u00ad\u2010165\t\r \u00a0(2002).\t\r \u00a044.\t\r \u00a0 F.\t\r \u00a0Garcia-\u00ad\u2010del\t\r \u00a0Portillo,\t\r \u00a0M.\t\r \u00a0G.\t\r \u00a0Pucciarelli,\t\r \u00a0W.\t\r \u00a0A.\t\r \u00a0Jefferies,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0induces\t\r \u00a0selective\t\r \u00a0aggregation\t\r \u00a0and\t\r \u00a0internalization\t\r \u00a0of\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0surface\t\r \u00a0proteins\t\r \u00a0during\t\r \u00a0invasion\t\r \u00a0of\t\r \u00a0epithelial\t\r \u00a0cells.\t\r \u00a0J\t\r \u00a0Cell\t\r \u00a0Sci\t\r \u00a0107\t\r \u00a0(\t\r \u00a0Pt\t\r \u00a07),\t\r \u00a02005-\u00ad\u20102020\t\r \u00a0(1994).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 146\t\r \u00a0 45.\t\r \u00a0 J.\t\r \u00a0C.\t\r \u00a0Patel,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Differential\t\r \u00a0activation\t\r \u00a0and\t\r \u00a0function\t\r \u00a0of\t\r \u00a0Rho\t\r \u00a0GTPases\t\r \u00a0during\t\r \u00a0Salmonella-\u00ad\u2010host\t\r \u00a0cell\t\r \u00a0interactions.\t\r \u00a0J\t\r \u00a0Cell\t\r \u00a0Biol\t\r \u00a0175,\t\r \u00a0453-\u00ad\u2010463\t\r \u00a0(2006).\t\r \u00a046.\t\r \u00a0 A.\t\r \u00a0K.\t\r \u00a0Criss,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Casanova,\t\r \u00a0Coordinate\t\r \u00a0regulation\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0Typhimurium\t\r \u00a0invasion\t\r \u00a0of\t\r \u00a0epithelial\t\r \u00a0cells\t\r \u00a0by\t\r \u00a0the\t\r \u00a0Arp2\/3\t\r \u00a0complex\t\r \u00a0and\t\r \u00a0Rho\t\r \u00a0GTPases.\t\r \u00a0Infect\t\r \u00a0Immun\t\r \u00a071,\t\r \u00a02885-\u00ad\u20102891\t\r \u00a0(2003).\t\r \u00a047.\t\r \u00a0 K.\t\r \u00a0E.\t\r \u00a0Unsworth,\t\r \u00a0M.\t\r \u00a0Way,\t\r \u00a0M.\t\r \u00a0McNiven,\t\r \u00a0L.\t\r \u00a0Machesky,\t\r \u00a0D.\t\r \u00a0W.\t\r \u00a0Holden,\t\r \u00a0Analysis\t\r \u00a0of\t\r \u00a0the\t\r \u00a0mechanisms\t\r \u00a0of\t\r \u00a0Salmonella-\u00ad\u2010induced\t\r \u00a0actin\t\r \u00a0assembly\t\r \u00a0during\t\r \u00a0invasion\t\r \u00a0of\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0and\t\r \u00a0intracellular\t\r \u00a0replication.\t\r \u00a0Cell\t\r \u00a0Microbiol\t\r \u00a06,\t\r \u00a01041-\u00ad\u20101055\t\r \u00a0(2004).\t\r \u00a048.\t\r \u00a0 J.\t\r \u00a0Shi,\t\r \u00a0G.\t\r \u00a0Scita,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Casanova,\t\r \u00a0WAVE2\t\r \u00a0signaling\t\r \u00a0mediates\t\r \u00a0invasion\t\r \u00a0of\t\r \u00a0polarized\t\r \u00a0epithelial\t\r \u00a0cells\t\r \u00a0by\t\r \u00a0Salmonella\t\r \u00a0typhimurium.\t\r \u00a0J\t\r \u00a0Biol\t\r \u00a0Chem\t\r \u00a0280,\t\r \u00a029849-\u00ad\u201029855\t\r \u00a0(2005).\t\r \u00a049.\t\r \u00a0 M.\t\r \u00a0R.\t\r \u00a0Terebiznik,\t\r \u00a0O.\t\r \u00a0V.\t\r \u00a0Vieira,\t\r \u00a0S.\t\r \u00a0L.\t\r \u00a0Marcus,\t\r \u00a0A.\t\r \u00a0Slade,\t\r \u00a0C.\t\r \u00a0M.\t\r \u00a0Yip,\t\r \u00a0W.\t\r \u00a0S.\t\r \u00a0Trimble,\t\r \u00a0T.\t\r \u00a0Meyer,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0S.\t\r \u00a0Grinstein,\t\r \u00a0Elimination\t\r \u00a0of\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0PtdIns(4,5)P(2)\t\r \u00a0by\t\r \u00a0bacterial\t\r \u00a0SigD\t\r \u00a0promotes\t\r \u00a0membrane\t\r \u00a0fission\t\r \u00a0during\t\r \u00a0invasion\t\r \u00a0by\t\r \u00a0Salmonella.\t\r \u00a0 Nat\t\r \u00a0Cell\t\r \u00a0Biol\t\r \u00a04,\t\r \u00a0766-\u00ad\u2010773\t\r \u00a0(2002).\t\r \u00a050.\t\r \u00a0 S.\t\r \u00a0Dai,\t\r \u00a0Y.\t\r \u00a0Zhang,\t\r \u00a0T.\t\r \u00a0Weimbs,\t\r \u00a0M.\t\r \u00a0B.\t\r \u00a0Yaffe,\t\r \u00a0D.\t\r \u00a0Zhou,\t\r \u00a0Bacteria-\u00ad\u2010generated\t\r \u00a0PtdIns(3)P\t\r \u00a0recruits\t\r \u00a0VAMP8\t\r \u00a0to\t\r \u00a0facilitate\t\r \u00a0phagocytosis.\t\r \u00a0Traffic\t\r \u00a08,\t\r \u00a01365-\u00ad\u20101374\t\r \u00a0(2007).\t\r \u00a051.\t\r \u00a0 J.\t\r \u00a0P.\t\r \u00a0Gorvel,\t\r \u00a0S.\t\r \u00a0Meresse,\t\r \u00a0Maturation\t\r \u00a0steps\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Salmonella-\u00ad\u2010containing\t\r \u00a0vacuole.\t\r \u00a0Microbes\t\r \u00a0Infect\t\r \u00a03,\t\r \u00a01299-\u00ad\u20101303\t\r \u00a0(2001).\t\r \u00a052.\t\r \u00a0 J.\t\r \u00a0H.\t\r \u00a0Brumell,\t\r \u00a0S.\t\r \u00a0Grinstein,\t\r \u00a0Salmonella\t\r \u00a0redirects\t\r \u00a0phagosomal\t\r \u00a0maturation.\t\r \u00a0Curr\t\r \u00a0 Opin\t\r \u00a0Microbiol\t\r \u00a07,\t\r \u00a078-\u00ad\u201084\t\r \u00a0(2004).\t\r \u00a053.\t\r \u00a0 O.\t\r \u00a0Steele-\u00ad\u2010Mortimer,\t\r \u00a0The\t\r \u00a0Salmonella-\u00ad\u2010containing\t\r \u00a0vacuole:\t\r \u00a0moving\t\r \u00a0with\t\r \u00a0the\t\r \u00a0times.\t\r \u00a0Curr\t\r \u00a0Opin\t\r \u00a0Microbiol\t\r \u00a011,\t\r \u00a038-\u00ad\u201045\t\r \u00a0(2008).\t\r \u00a054.\t\r \u00a0 O.\t\r \u00a0Steele-\u00ad\u2010Mortimer,\t\r \u00a0S.\t\r \u00a0Meresse,\t\r \u00a0J.\t\r \u00a0P.\t\r \u00a0Gorvel,\t\r \u00a0B.\t\r \u00a0H.\t\r \u00a0Toh,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0Biogenesis\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0typhimurium-\u00ad\u2010containing\t\r \u00a0vacuoles\t\r \u00a0in\t\r \u00a0epithelial\t\r \u00a0cells\t\r \u00a0involves\t\r \u00a0interactions\t\r \u00a0with\t\r \u00a0the\t\r \u00a0early\t\r \u00a0endocytic\t\r \u00a0pathway.\t\r \u00a0Cell\t\r \u00a0Microbiol\t\r \u00a01,\t\r \u00a033-\u00ad\u201049\t\r \u00a0(1999).\t\r \u00a055.\t\r \u00a0 K.\t\r \u00a0Mukherjee,\t\r \u00a0S.\t\r \u00a0A.\t\r \u00a0Siddiqi,\t\r \u00a0S.\t\r \u00a0Hashim,\t\r \u00a0M.\t\r \u00a0Raje,\t\r \u00a0S.\t\r \u00a0K.\t\r \u00a0Basu,\t\r \u00a0A.\t\r \u00a0Mukhopadhyay,\t\r \u00a0Live\t\r \u00a0Salmonella\t\r \u00a0recruits\t\r \u00a0N-\u00ad\u2010ethylmaleimide-\u00ad\u2010sensitive\t\r \u00a0fusion\t\r \u00a0protein\t\r \u00a0on\t\r \u00a0phagosomal\t\r \u00a0membrane\t\r \u00a0and\t\r \u00a0promotes\t\r \u00a0fusion\t\r \u00a0with\t\r \u00a0early\t\r \u00a0endosome.\t\r \u00a0J\t\r \u00a0Cell\t\r \u00a0Biol\t\r \u00a0 148,\t\r \u00a0741-\u00ad\u2010753\t\r \u00a0(2000).\t\r \u00a056.\t\r \u00a0 C.\t\r \u00a0C.\t\r \u00a0Scott,\t\r \u00a0P.\t\r \u00a0Cuellar-\u00ad\u2010Mata,\t\r \u00a0T.\t\r \u00a0Matsuo,\t\r \u00a0H.\t\r \u00a0W.\t\r \u00a0Davidson,\t\r \u00a0S.\t\r \u00a0Grinstein,\t\r \u00a0Role\t\r \u00a0of\t\r \u00a03-\u00ad\u2010phosphoinositides\t\r \u00a0in\t\r \u00a0the\t\r \u00a0maturation\t\r \u00a0of\t\r \u00a0Salmonella-\u00ad\u2010containing\t\r \u00a0vacuoles\t\r \u00a0within\t\r \u00a0host\t\r \u00a0cells.\t\r \u00a0J\t\r \u00a0Biol\t\r \u00a0Chem\t\r \u00a0277,\t\r \u00a012770-\u00ad\u201012776\t\r \u00a0(2002).\t\r \u00a057.\t\r \u00a0 L.\t\r \u00a0D.\t\r \u00a0Hernandez,\t\r \u00a0K.\t\r \u00a0Hueffer,\t\r \u00a0M.\t\r \u00a0R.\t\r \u00a0Wenk,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Salmonella\t\r \u00a0modulates\t\r \u00a0vesicular\t\r \u00a0traffic\t\r \u00a0by\t\r \u00a0altering\t\r \u00a0phosphoinositide\t\r \u00a0metabolism.\t\r \u00a0Science\t\r \u00a0304,\t\r \u00a01805-\u00ad\u20101807\t\r \u00a0(2004).\t\r \u00a058.\t\r \u00a0 G.\t\r \u00a0V.\t\r \u00a0Mallo,\t\r \u00a0M.\t\r \u00a0Espina,\t\r \u00a0A.\t\r \u00a0C.\t\r \u00a0Smith,\t\r \u00a0M.\t\r \u00a0R.\t\r \u00a0Terebiznik,\t\r \u00a0A.\t\r \u00a0Aleman,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0L.\t\r \u00a0E.\t\r \u00a0Rameh,\t\r \u00a0S.\t\r \u00a0Grinstein,\t\r \u00a0J.\t\r \u00a0H.\t\r \u00a0Brumell,\t\r \u00a0SopB\t\r \u00a0promotes\t\r \u00a0phosphatidylinositol\t\r \u00a03-\u00ad\u2010phosphate\t\r \u00a0formation\t\r \u00a0on\t\r \u00a0Salmonella\t\r \u00a0vacuoles\t\r \u00a0by\t\r \u00a0recruiting\t\r \u00a0Rab5\t\r \u00a0and\t\r \u00a0Vps34.\t\r \u00a0J\t\r \u00a0 Cell\t\r \u00a0Biol\t\r \u00a0182,\t\r \u00a0741-\u00ad\u2010752\t\r \u00a0(2008).\t\r \u00a059.\t\r \u00a0 M.\t\r \u00a0V.\t\r \u00a0Bujny,\t\r \u00a0P.\t\r \u00a0A.\t\r \u00a0Ewels,\t\r \u00a0S.\t\r \u00a0Humphrey,\t\r \u00a0N.\t\r \u00a0Attar,\t\r \u00a0M.\t\r \u00a0A.\t\r \u00a0Jepson,\t\r \u00a0P.\t\r \u00a0J.\t\r \u00a0Cullen,\t\r \u00a0Sorting\t\r \u00a0nexin-\u00ad\u20101\t\r \u00a0defines\t\r \u00a0an\t\r \u00a0early\t\r \u00a0phase\t\r \u00a0of\t\r \u00a0Salmonella-\u00ad\u2010containing\t\r \u00a0vacuole-\u00ad\u2010remodeling\t\r \u00a0during\t\r \u00a0Salmonella\t\r \u00a0infection.\t\r \u00a0J\t\r \u00a0Cell\t\r \u00a0Sci\t\r \u00a0121,\t\r \u00a02027-\u00ad\u20102036\t\r \u00a0(2008).\t\r \u00a060.\t\r \u00a0 V.\t\r \u00a0Braun,\t\r \u00a0A.\t\r \u00a0Wong,\t\r \u00a0M.\t\r \u00a0Landekic,\t\r \u00a0W.\t\r \u00a0J.\t\r \u00a0Hong,\t\r \u00a0S.\t\r \u00a0Grinstein,\t\r \u00a0J.\t\r \u00a0H.\t\r \u00a0Brumell,\t\r \u00a0Sorting\t\r \u00a0nexin\t\r \u00a03\t\r \u00a0(SNX3)\t\r \u00a0is\t\r \u00a0a\t\r \u00a0component\t\r \u00a0of\t\r \u00a0a\t\r \u00a0tubular\t\r \u00a0endosomal\t\r \u00a0network\t\r \u00a0induced\t\r \u00a0by\t\r \u00a0 \t\r \u00a0\t\r \u00a0 147\t\r \u00a0 Salmonella\t\r \u00a0and\t\r \u00a0involved\t\r \u00a0in\t\r \u00a0maturation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Salmonella-\u00ad\u2010containing\t\r \u00a0vacuole.\t\r \u00a0 Cell\t\r \u00a0Microbiol\t\r \u00a012,\t\r \u00a01352-\u00ad\u20101367\t\r \u00a0(2010).\t\r \u00a061.\t\r \u00a0 A.\t\r \u00a0C.\t\r \u00a0Smith,\t\r \u00a0J.\t\r \u00a0T.\t\r \u00a0Cirulis,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Casanova,\t\r \u00a0M.\t\r \u00a0A.\t\r \u00a0Scidmore,\t\r \u00a0J.\t\r \u00a0H.\t\r \u00a0Brumell,\t\r \u00a0Interaction\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Salmonella-\u00ad\u2010containing\t\r \u00a0vacuole\t\r \u00a0with\t\r \u00a0the\t\r \u00a0endocytic\t\r \u00a0recycling\t\r \u00a0system.\t\r \u00a0J\t\r \u00a0Biol\t\r \u00a0Chem\t\r \u00a0280,\t\r \u00a024634-\u00ad\u201024641\t\r \u00a0(2005).\t\r \u00a062.\t\r \u00a0 F.\t\r \u00a0Garcia-\u00ad\u2010del\t\r \u00a0Portillo,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0Targeting\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0to\t\r \u00a0vesicles\t\r \u00a0containing\t\r \u00a0lysosomal\t\r \u00a0membrane\t\r \u00a0glycoproteins\t\r \u00a0bypasses\t\r \u00a0compartments\t\r \u00a0with\t\r \u00a0mannose\t\r \u00a06-\u00ad\u2010phosphate\t\r \u00a0receptors.\t\r \u00a0J\t\r \u00a0Cell\t\r \u00a0Biol\t\r \u00a0129,\t\r \u00a081-\u00ad\u201097\t\r \u00a0(1995).\t\r \u00a063.\t\r \u00a0 S.\t\r \u00a0Meresse,\t\r \u00a0O.\t\r \u00a0Steele-\u00ad\u2010Mortimer,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0J.\t\r \u00a0P.\t\r \u00a0Gorvel,\t\r \u00a0The\t\r \u00a0rab7\t\r \u00a0GTPase\t\r \u00a0controls\t\r \u00a0the\t\r \u00a0maturation\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0typhimurium-\u00ad\u2010containing\t\r \u00a0vacuoles\t\r \u00a0in\t\r \u00a0HeLa\t\r \u00a0cells.\t\r \u00a0EMBO\t\r \u00a0J\t\r \u00a018,\t\r \u00a04394-\u00ad\u20104403\t\r \u00a0(1999).\t\r \u00a064.\t\r \u00a0 M.\t\r \u00a0C.\t\r \u00a0Kerr,\t\r \u00a0J.\t\r \u00a0T.\t\r \u00a0Wang,\t\r \u00a0N.\t\r \u00a0A.\t\r \u00a0Castro,\t\r \u00a0N.\t\r \u00a0A.\t\r \u00a0Hamilton,\t\r \u00a0L.\t\r \u00a0Town,\t\r \u00a0D.\t\r \u00a0L.\t\r \u00a0Brown,\t\r \u00a0F.\t\r \u00a0A.\t\r \u00a0Meunier,\t\r \u00a0N.\t\r \u00a0F.\t\r \u00a0Brown,\t\r \u00a0J.\t\r \u00a0L.\t\r \u00a0Stow,\t\r \u00a0R.\t\r \u00a0D.\t\r \u00a0Teasdale,\t\r \u00a0Inhibition\t\r \u00a0of\t\r \u00a0the\t\r \u00a0PtdIns(5)\t\r \u00a0kinase\t\r \u00a0PIKfyve\t\r \u00a0disrupts\t\r \u00a0intracellular\t\r \u00a0replication\t\r \u00a0of\t\r \u00a0Salmonella.\t\r \u00a0EMBO\t\r \u00a0J\t\r \u00a029,\t\r \u00a01331-\u00ad\u20101347\t\r \u00a0(2010).\t\r \u00a065.\t\r \u00a0 A.\t\r \u00a0C.\t\r \u00a0Smith,\t\r \u00a0W.\t\r \u00a0D.\t\r \u00a0Heo,\t\r \u00a0V.\t\r \u00a0Braun,\t\r \u00a0X.\t\r \u00a0Jiang,\t\r \u00a0C.\t\r \u00a0Macrae,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Casanova,\t\r \u00a0M.\t\r \u00a0A.\t\r \u00a0Scidmore,\t\r \u00a0S.\t\r \u00a0Grinstein,\t\r \u00a0T.\t\r \u00a0Meyer,\t\r \u00a0J.\t\r \u00a0H.\t\r \u00a0Brumell,\t\r \u00a0A\t\r \u00a0network\t\r \u00a0of\t\r \u00a0Rab\t\r \u00a0GTPases\t\r \u00a0controls\t\r \u00a0phagosome\t\r \u00a0maturation\t\r \u00a0and\t\r \u00a0is\t\r \u00a0modulated\t\r \u00a0by\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0Typhimurium.\t\r \u00a0J\t\r \u00a0Cell\t\r \u00a0Biol\t\r \u00a0176,\t\r \u00a0263-\u00ad\u2010268\t\r \u00a0(2007).\t\r \u00a066.\t\r \u00a0 C.\t\r \u00a0Kuijl,\t\r \u00a0N.\t\r \u00a0D.\t\r \u00a0Savage,\t\r \u00a0M.\t\r \u00a0Marsman,\t\r \u00a0A.\t\r \u00a0W.\t\r \u00a0Tuin,\t\r \u00a0L.\t\r \u00a0Janssen,\t\r \u00a0D.\t\r \u00a0A.\t\r \u00a0Egan,\t\r \u00a0M.\t\r \u00a0Ketema,\t\r \u00a0R.\t\r \u00a0van\t\r \u00a0den\t\r \u00a0Nieuwendijk,\t\r \u00a0S.\t\r \u00a0J.\t\r \u00a0van\t\r \u00a0den\t\r \u00a0Eeden,\t\r \u00a0A.\t\r \u00a0Geluk,\t\r \u00a0A.\t\r \u00a0Poot,\t\r \u00a0G.\t\r \u00a0van\t\r \u00a0der\t\r \u00a0Marel,\t\r \u00a0R.\t\r \u00a0L.\t\r \u00a0Beijersbergen,\t\r \u00a0H.\t\r \u00a0Overkleeft,\t\r \u00a0T.\t\r \u00a0H.\t\r \u00a0Ottenhoff,\t\r \u00a0J.\t\r \u00a0Neefjes,\t\r \u00a0Intracellular\t\r \u00a0bacterial\t\r \u00a0growth\t\r \u00a0is\t\r \u00a0controlled\t\r \u00a0by\t\r \u00a0a\t\r \u00a0kinase\t\r \u00a0network\t\r \u00a0around\t\r \u00a0PKB\/AKT1.\t\r \u00a0Nature\t\r \u00a0450,\t\r \u00a0725-\u00ad\u2010730\t\r \u00a0(2007).\t\r \u00a067.\t\r \u00a0 J.\t\r \u00a0Guignot,\t\r \u00a0E.\t\r \u00a0Caron,\t\r \u00a0C.\t\r \u00a0Beuzon,\t\r \u00a0C.\t\r \u00a0Bucci,\t\r \u00a0J.\t\r \u00a0Kagan,\t\r \u00a0C.\t\r \u00a0Roy,\t\r \u00a0D.\t\r \u00a0W.\t\r \u00a0Holden,\t\r \u00a0Microtubule\t\r \u00a0motors\t\r \u00a0control\t\r \u00a0membrane\t\r \u00a0dynamics\t\r \u00a0of\t\r \u00a0Salmonella-\u00ad\u2010containing\t\r \u00a0vacuoles.\t\r \u00a0J\t\r \u00a0Cell\t\r \u00a0Sci\t\r \u00a0117,\t\r \u00a01033-\u00ad\u20101045\t\r \u00a0(2004).\t\r \u00a068.\t\r \u00a0 R.\t\r \u00a0E.\t\r \u00a0Harrison,\t\r \u00a0J.\t\r \u00a0H.\t\r \u00a0Brumell,\t\r \u00a0A.\t\r \u00a0Khandani,\t\r \u00a0C.\t\r \u00a0Bucci,\t\r \u00a0C.\t\r \u00a0C.\t\r \u00a0Scott,\t\r \u00a0X.\t\r \u00a0Jiang,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0S.\t\r \u00a0Grinstein,\t\r \u00a0Salmonella\t\r \u00a0impairs\t\r \u00a0RILP\t\r \u00a0recruitment\t\r \u00a0to\t\r \u00a0Rab7\t\r \u00a0during\t\r \u00a0maturation\t\r \u00a0of\t\r \u00a0invasion\t\r \u00a0vacuoles.\t\r \u00a0Mol\t\r \u00a0Biol\t\r \u00a0Cell\t\r \u00a015,\t\r \u00a03146-\u00ad\u20103154\t\r \u00a0(2004).\t\r \u00a069.\t\r \u00a0 M.\t\r \u00a0Marsman,\t\r \u00a0I.\t\r \u00a0Jordens,\t\r \u00a0C.\t\r \u00a0Kuijl,\t\r \u00a0L.\t\r \u00a0Janssen,\t\r \u00a0J.\t\r \u00a0Neefjes,\t\r \u00a0Dynein-\u00ad\u2010mediated\t\r \u00a0vesicle\t\r \u00a0transport\t\r \u00a0controls\t\r \u00a0intracellular\t\r \u00a0Salmonella\t\r \u00a0replication.\t\r \u00a0Mol\t\r \u00a0Biol\t\r \u00a0Cell\t\r \u00a0 15,\t\r \u00a02954-\u00ad\u20102964\t\r \u00a0(2004).\t\r \u00a070.\t\r \u00a0 J.\t\r \u00a0A.\t\r \u00a0Wasylnka,\t\r \u00a0M.\t\r \u00a0A.\t\r \u00a0Bakowski,\t\r \u00a0J.\t\r \u00a0Szeto,\t\r \u00a0M.\t\r \u00a0B.\t\r \u00a0Ohlson,\t\r \u00a0W.\t\r \u00a0S.\t\r \u00a0Trimble,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0J.\t\r \u00a0H.\t\r \u00a0Brumell,\t\r \u00a0Role\t\r \u00a0for\t\r \u00a0myosin\t\r \u00a0II\t\r \u00a0in\t\r \u00a0regulating\t\r \u00a0positioning\t\r \u00a0of\t\r \u00a0Salmonella-\u00ad\u2010containing\t\r \u00a0vacuoles\t\r \u00a0and\t\r \u00a0intracellular\t\r \u00a0replication.\t\r \u00a0Infect\t\r \u00a0Immun\t\r \u00a076,\t\r \u00a02722-\u00ad\u20102735\t\r \u00a0(2008).\t\r \u00a071.\t\r \u00a0 F.\t\r \u00a0Garcia-\u00ad\u2010del\t\r \u00a0Portillo,\t\r \u00a0M.\t\r \u00a0B.\t\r \u00a0Zwick,\t\r \u00a0K.\t\r \u00a0Y.\t\r \u00a0Leung,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0Salmonella\t\r \u00a0induces\t\r \u00a0the\t\r \u00a0formation\t\r \u00a0of\t\r \u00a0filamentous\t\r \u00a0structures\t\r \u00a0containing\t\r \u00a0lysosomal\t\r \u00a0membrane\t\r \u00a0glycoproteins\t\r \u00a0in\t\r \u00a0epithelial\t\r \u00a0cells.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a090,\t\r \u00a010544-\u00ad\u201010548\t\r \u00a0(1993).\t\r \u00a072.\t\r \u00a0 L.\t\r \u00a0A.\t\r \u00a0Knodler,\t\r \u00a0B.\t\r \u00a0A.\t\r \u00a0Vallance,\t\r \u00a0M.\t\r \u00a0Hensel,\t\r \u00a0D.\t\r \u00a0Jackel,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0O.\t\r \u00a0Steele-\u00ad\u2010Mortimer,\t\r \u00a0Salmonella\t\r \u00a0type\t\r \u00a0III\t\r \u00a0effectors\t\r \u00a0PipB\t\r \u00a0and\t\r \u00a0PipB2\t\r \u00a0are\t\r \u00a0targeted\t\r \u00a0to\t\r \u00a0detergent-\u00ad\u2010resistant\t\r \u00a0microdomains\t\r \u00a0on\t\r \u00a0internal\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0membranes.\t\r \u00a0Mol\t\r \u00a0 Microbiol\t\r \u00a049,\t\r \u00a0685-\u00ad\u2010704\t\r \u00a0(2003).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 148\t\r \u00a0 73.\t\r \u00a0 R.\t\r \u00a0Rajashekar,\t\r \u00a0D.\t\r \u00a0Liebl,\t\r \u00a0A.\t\r \u00a0Seitz,\t\r \u00a0M.\t\r \u00a0Hensel,\t\r \u00a0Dynamic\t\r \u00a0remodeling\t\r \u00a0of\t\r \u00a0the\t\r \u00a0endosomal\t\r \u00a0system\t\r \u00a0during\t\r \u00a0formation\t\r \u00a0of\t\r \u00a0Salmonella-\u00ad\u2010induced\t\r \u00a0filaments\t\r \u00a0by\t\r \u00a0intracellular\t\r \u00a0Salmonella\t\r \u00a0enterica.\t\r \u00a0Traffic\t\r \u00a09,\t\r \u00a02100-\u00ad\u20102116\t\r \u00a0(2008).\t\r \u00a074.\t\r \u00a0 D.\t\r \u00a0Drecktrah,\t\r \u00a0S.\t\r \u00a0Levine-\u00ad\u2010Wilkinson,\t\r \u00a0T.\t\r \u00a0Dam,\t\r \u00a0S.\t\r \u00a0Winfree,\t\r \u00a0L.\t\r \u00a0A.\t\r \u00a0Knodler,\t\r \u00a0T.\t\r \u00a0A.\t\r \u00a0Schroer,\t\r \u00a0O.\t\r \u00a0Steele-\u00ad\u2010Mortimer,\t\r \u00a0Dynamic\t\r \u00a0behavior\t\r \u00a0of\t\r \u00a0Salmonella-\u00ad\u2010induced\t\r \u00a0membrane\t\r \u00a0tubules\t\r \u00a0in\t\r \u00a0epithelial\t\r \u00a0cells.\t\r \u00a0Traffic\t\r \u00a09,\t\r \u00a02117-\u00ad\u20102129\t\r \u00a0(2008).\t\r \u00a075.\t\r \u00a0 C.\t\r \u00a0L.\t\r \u00a0Birmingham,\t\r \u00a0X.\t\r \u00a0Jiang,\t\r \u00a0M.\t\r \u00a0B.\t\r \u00a0Ohlson,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0J.\t\r \u00a0H.\t\r \u00a0Brumell,\t\r \u00a0Salmonella-\u00ad\u2010induced\t\r \u00a0filament\t\r \u00a0formation\t\r \u00a0is\t\r \u00a0a\t\r \u00a0dynamic\t\r \u00a0phenotype\t\r \u00a0induced\t\r \u00a0by\t\r \u00a0rapidly\t\r \u00a0replicating\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0typhimurium\t\r \u00a0in\t\r \u00a0epithelial\t\r \u00a0cells.\t\r \u00a0Infect\t\r \u00a0 Immun\t\r \u00a073,\t\r \u00a01204-\u00ad\u20101208\t\r \u00a0(2005).\t\r \u00a076.\t\r \u00a0 J.\t\r \u00a0H.\t\r \u00a0Brumell,\t\r \u00a0P.\t\r \u00a0Tang,\t\r \u00a0S.\t\r \u00a0D.\t\r \u00a0Mills,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0Characterization\t\r \u00a0of\t\r \u00a0Salmonella-\u00ad\u2010induced\t\r \u00a0filaments\t\r \u00a0(Sifs)\t\r \u00a0reveals\t\r \u00a0a\t\r \u00a0delayed\t\r \u00a0interaction\t\r \u00a0between\t\r \u00a0Salmonella-\u00ad\u2010containing\t\r \u00a0vacuoles\t\r \u00a0and\t\r \u00a0late\t\r \u00a0endocytic\t\r \u00a0compartments.\t\r \u00a0Traffic\t\r \u00a02,\t\r \u00a0643-\u00ad\u2010653\t\r \u00a0(2001).\t\r \u00a077.\t\r \u00a0 L.\t\r \u00a0J.\t\r \u00a0Mota,\t\r \u00a0A.\t\r \u00a0E.\t\r \u00a0Ramsden,\t\r \u00a0M.\t\r \u00a0Liu,\t\r \u00a0J.\t\r \u00a0D.\t\r \u00a0Castle,\t\r \u00a0D.\t\r \u00a0W.\t\r \u00a0Holden,\t\r \u00a0SCAMP3\t\r \u00a0is\t\r \u00a0a\t\r \u00a0component\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Salmonella-\u00ad\u2010induced\t\r \u00a0tubular\t\r \u00a0network\t\r \u00a0and\t\r \u00a0reveals\t\r \u00a0an\t\r \u00a0interaction\t\r \u00a0between\t\r \u00a0bacterial\t\r \u00a0effectors\t\r \u00a0and\t\r \u00a0post-\u00ad\u2010Golgi\t\r \u00a0trafficking.\t\r \u00a0Cell\t\r \u00a0 Microbiol\t\r \u00a011,\t\r \u00a01236-\u00ad\u20101253\t\r \u00a0(2009).\t\r \u00a078.\t\r \u00a0 V.\t\r \u00a0Kuhle,\t\r \u00a0G.\t\r \u00a0L.\t\r \u00a0Abrahams,\t\r \u00a0M.\t\r \u00a0Hensel,\t\r \u00a0Intracellular\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0redirect\t\r \u00a0exocytic\t\r \u00a0transport\t\r \u00a0processes\t\r \u00a0in\t\r \u00a0a\t\r \u00a0Salmonella\t\r \u00a0pathogenicity\t\r \u00a0island\t\r \u00a02-\u00ad\u2010dependent\t\r \u00a0manner.\t\r \u00a0Traffic\t\r \u00a07,\t\r \u00a0716-\u00ad\u2010730\t\r \u00a0(2006).\t\r \u00a079.\t\r \u00a0 D.\t\r \u00a0Lombardi,\t\r \u00a0T.\t\r \u00a0Soldati,\t\r \u00a0M.\t\r \u00a0A.\t\r \u00a0Riederer,\t\r \u00a0Y.\t\r \u00a0Goda,\t\r \u00a0M.\t\r \u00a0Zerial,\t\r \u00a0S.\t\r \u00a0R.\t\r \u00a0Pfeffer,\t\r \u00a0Rab9\t\r \u00a0functions\t\r \u00a0in\t\r \u00a0transport\t\r \u00a0between\t\r \u00a0late\t\r \u00a0endosomes\t\r \u00a0and\t\r \u00a0the\t\r \u00a0trans\t\r \u00a0Golgi\t\r \u00a0network.\t\r \u00a0 EMBO\t\r \u00a0J\t\r \u00a012,\t\r \u00a0677-\u00ad\u2010682\t\r \u00a0(1993).\t\r \u00a080.\t\r \u00a0 E.\t\r \u00a0Boucrot,\t\r \u00a0T.\t\r \u00a0Henry,\t\r \u00a0J.\t\r \u00a0P.\t\r \u00a0Borg,\t\r \u00a0J.\t\r \u00a0P.\t\r \u00a0Gorvel,\t\r \u00a0S.\t\r \u00a0Meresse,\t\r \u00a0The\t\r \u00a0intracellular\t\r \u00a0fate\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0depends\t\r \u00a0on\t\r \u00a0the\t\r \u00a0recruitment\t\r \u00a0of\t\r \u00a0kinesin.\t\r \u00a0Science\t\r \u00a0308,\t\r \u00a01174-\u00ad\u20101178\t\r \u00a0(2005).\t\r \u00a081.\t\r \u00a0 D.\t\r \u00a0M.\t\r \u00a0Catron,\t\r \u00a0M.\t\r \u00a0D.\t\r \u00a0Sylvester,\t\r \u00a0Y.\t\r \u00a0Lange,\t\r \u00a0M.\t\r \u00a0Kadekoppala,\t\r \u00a0B.\t\r \u00a0D.\t\r \u00a0Jones,\t\r \u00a0D.\t\r \u00a0M.\t\r \u00a0Monack,\t\r \u00a0S.\t\r \u00a0Falkow,\t\r \u00a0K.\t\r \u00a0Haldar,\t\r \u00a0The\t\r \u00a0Salmonella-\u00ad\u2010containing\t\r \u00a0vacuole\t\r \u00a0is\t\r \u00a0a\t\r \u00a0major\t\r \u00a0site\t\r \u00a0of\t\r \u00a0intracellular\t\r \u00a0cholesterol\t\r \u00a0accumulation\t\r \u00a0and\t\r \u00a0recruits\t\r \u00a0the\t\r \u00a0GPI-\u00ad\u2010anchored\t\r \u00a0protein\t\r \u00a0CD55.\t\r \u00a0Cell\t\r \u00a0Microbiol\t\r \u00a04,\t\r \u00a0315-\u00ad\u2010328\t\r \u00a0(2002).\t\r \u00a082.\t\r \u00a0 D.\t\r \u00a0M.\t\r \u00a0Catron,\t\r \u00a0Y.\t\r \u00a0Lange,\t\r \u00a0J.\t\r \u00a0Borensztajn,\t\r \u00a0M.\t\r \u00a0D.\t\r \u00a0Sylvester,\t\r \u00a0B.\t\r \u00a0D.\t\r \u00a0Jones,\t\r \u00a0K.\t\r \u00a0Haldar,\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0Typhimurium\t\r \u00a0requires\t\r \u00a0nonsterol\t\r \u00a0precursors\t\r \u00a0of\t\r \u00a0the\t\r \u00a0cholesterol\t\r \u00a0biosynthetic\t\r \u00a0pathway\t\r \u00a0for\t\r \u00a0intracellular\t\r \u00a0proliferation.\t\r \u00a0Infect\t\r \u00a0 Immun\t\r \u00a072,\t\r \u00a01036-\u00ad\u20101042\t\r \u00a0(2004).\t\r \u00a083.\t\r \u00a0 S.\t\r \u00a0Meresse,\t\r \u00a0K.\t\r \u00a0E.\t\r \u00a0Unsworth,\t\r \u00a0A.\t\r \u00a0Habermann,\t\r \u00a0G.\t\r \u00a0Griffiths,\t\r \u00a0F.\t\r \u00a0Fang,\t\r \u00a0M.\t\r \u00a0J.\t\r \u00a0Martinez-\u00ad\u2010Lorenzo,\t\r \u00a0S.\t\r \u00a0R.\t\r \u00a0Waterman,\t\r \u00a0J.\t\r \u00a0P.\t\r \u00a0Gorvel,\t\r \u00a0D.\t\r \u00a0W.\t\r \u00a0Holden,\t\r \u00a0Remodelling\t\r \u00a0of\t\r \u00a0the\t\r \u00a0actin\t\r \u00a0cytoskeleton\t\r \u00a0is\t\r \u00a0essential\t\r \u00a0for\t\r \u00a0replication\t\r \u00a0of\t\r \u00a0intravacuolar\t\r \u00a0Salmonella.\t\r \u00a0Cell\t\r \u00a0 Microbiol\t\r \u00a03,\t\r \u00a0567-\u00ad\u2010577\t\r \u00a0(2001).\t\r \u00a084.\t\r \u00a0 P.\t\r \u00a0J.\t\r \u00a0Sansonetti,\t\r \u00a0D.\t\r \u00a0J.\t\r \u00a0Kopecko,\t\r \u00a0S.\t\r \u00a0B.\t\r \u00a0Formal,\t\r \u00a0Involvement\t\r \u00a0of\t\r \u00a0a\t\r \u00a0plasmid\t\r \u00a0in\t\r \u00a0the\t\r \u00a0invasive\t\r \u00a0ability\t\r \u00a0of\t\r \u00a0Shigella\t\r \u00a0flexneri.\t\r \u00a0Infect\t\r \u00a0Immun\t\r \u00a035,\t\r \u00a0852-\u00ad\u2010860\t\r \u00a0(1982).\t\r \u00a085.\t\r \u00a0 E.\t\r \u00a0A.\t\r \u00a0Groisman,\t\r \u00a0H.\t\r \u00a0Ochman,\t\r \u00a0How\t\r \u00a0Salmonella\t\r \u00a0became\t\r \u00a0a\t\r \u00a0pathogen.\t\r \u00a0Trends\t\r \u00a0 Microbiol\t\r \u00a05,\t\r \u00a0343-\u00ad\u2010349\t\r \u00a0(1997).\t\r \u00a086.\t\r \u00a0 B.\t\r \u00a0D.\t\r \u00a0Jones,\t\r \u00a0S.\t\r \u00a0Falkow,\t\r \u00a0Salmonellosis:\t\r \u00a0host\t\r \u00a0immune\t\r \u00a0responses\t\r \u00a0and\t\r \u00a0bacterial\t\r \u00a0virulence\t\r \u00a0determinants.\t\r \u00a0Annu\t\r \u00a0Rev\t\r \u00a0Immunol\t\r \u00a014,\t\r \u00a0533-\u00ad\u2010561\t\r \u00a0(1996).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 149\t\r \u00a0 87.\t\r \u00a0 T.\t\r \u00a0K.\t\r \u00a0McDaniel,\t\r \u00a0K.\t\r \u00a0G.\t\r \u00a0Jarvis,\t\r \u00a0M.\t\r \u00a0S.\t\r \u00a0Donnenberg,\t\r \u00a0J.\t\r \u00a0B.\t\r \u00a0Kaper,\t\r \u00a0A\t\r \u00a0genetic\t\r \u00a0locus\t\r \u00a0of\t\r \u00a0enterocyte\t\r \u00a0effacement\t\r \u00a0conserved\t\r \u00a0among\t\r \u00a0diverse\t\r \u00a0enterobacterial\t\r \u00a0pathogens.\t\r \u00a0 Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a092,\t\r \u00a01664-\u00ad\u20101668\t\r \u00a0(1995).\t\r \u00a088.\t\r \u00a0 J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Molecular\t\r \u00a0genetic\t\r \u00a0bases\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0entry\t\r \u00a0into\t\r \u00a0host\t\r \u00a0cells.\t\r \u00a0Mol\t\r \u00a0 Microbiol\t\r \u00a020,\t\r \u00a0263-\u00ad\u2010271\t\r \u00a0(1996).\t\r \u00a089.\t\r \u00a0 J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Interaction\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0with\t\r \u00a0host\t\r \u00a0cells\t\r \u00a0through\t\r \u00a0the\t\r \u00a0centisome\t\r \u00a063\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0system.\t\r \u00a0Curr\t\r \u00a0Opin\t\r \u00a0Microbiol\t\r \u00a02,\t\r \u00a046-\u00ad\u201050\t\r \u00a0(1999).\t\r \u00a090.\t\r \u00a0 S.\t\r \u00a0Hapfelmeier,\t\r \u00a0B.\t\r \u00a0Stecher,\t\r \u00a0M.\t\r \u00a0Barthel,\t\r \u00a0M.\t\r \u00a0Kremer,\t\r \u00a0A.\t\r \u00a0J.\t\r \u00a0Muller,\t\r \u00a0M.\t\r \u00a0Heikenwalder,\t\r \u00a0T.\t\r \u00a0Stallmach,\t\r \u00a0M.\t\r \u00a0Hensel,\t\r \u00a0K.\t\r \u00a0Pfeffer,\t\r \u00a0S.\t\r \u00a0Akira,\t\r \u00a0W.\t\r \u00a0D.\t\r \u00a0Hardt,\t\r \u00a0The\t\r \u00a0Salmonella\t\r \u00a0pathogenicity\t\r \u00a0island\t\r \u00a0(SPI)-\u00ad\u20102\t\r \u00a0and\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0systems\t\r \u00a0allow\t\r \u00a0Salmonella\t\r \u00a0serovar\t\r \u00a0typhimurium\t\r \u00a0to\t\r \u00a0trigger\t\r \u00a0colitis\t\r \u00a0via\t\r \u00a0MyD88-\u00ad\u2010dependent\t\r \u00a0and\t\r \u00a0MyD88-\u00ad\u2010independent\t\r \u00a0mechanisms.\t\r \u00a0J\t\r \u00a0Immunol\t\r \u00a0174,\t\r \u00a01675-\u00ad\u20101685\t\r \u00a0(2005).\t\r \u00a091.\t\r \u00a0 B.\t\r \u00a0Coburn,\t\r \u00a0Y.\t\r \u00a0Li,\t\r \u00a0D.\t\r \u00a0Owen,\t\r \u00a0B.\t\r \u00a0A.\t\r \u00a0Vallance,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0Typhimurium\t\r \u00a0pathogenicity\t\r \u00a0island\t\r \u00a02\t\r \u00a0is\t\r \u00a0necessary\t\r \u00a0for\t\r \u00a0complete\t\r \u00a0virulence\t\r \u00a0in\t\r \u00a0a\t\r \u00a0mouse\t\r \u00a0model\t\r \u00a0of\t\r \u00a0infectious\t\r \u00a0enterocolitis.\t\r \u00a0Infect\t\r \u00a0Immun\t\r \u00a073,\t\r \u00a03219-\u00ad\u20103227\t\r \u00a0(2005).\t\r \u00a092.\t\r \u00a0 M.\t\r \u00a0Hensel,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Shea,\t\r \u00a0C.\t\r \u00a0Gleeson,\t\r \u00a0M.\t\r \u00a0D.\t\r \u00a0Jones,\t\r \u00a0E.\t\r \u00a0Dalton,\t\r \u00a0D.\t\r \u00a0W.\t\r \u00a0Holden,\t\r \u00a0Simultaneous\t\r \u00a0identification\t\r \u00a0of\t\r \u00a0bacterial\t\r \u00a0virulence\t\r \u00a0genes\t\r \u00a0by\t\r \u00a0negative\t\r \u00a0selection.\t\r \u00a0Science\t\r \u00a0269,\t\r \u00a0400-\u00ad\u2010403\t\r \u00a0(1995).\t\r \u00a093.\t\r \u00a0 J.\t\r \u00a0E.\t\r \u00a0Shea,\t\r \u00a0M.\t\r \u00a0Hensel,\t\r \u00a0C.\t\r \u00a0Gleeson,\t\r \u00a0D.\t\r \u00a0W.\t\r \u00a0Holden,\t\r \u00a0Identification\t\r \u00a0of\t\r \u00a0a\t\r \u00a0virulence\t\r \u00a0locus\t\r \u00a0encoding\t\r \u00a0a\t\r \u00a0second\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0system\t\r \u00a0in\t\r \u00a0Salmonella\t\r \u00a0typhimurium.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a093,\t\r \u00a02593-\u00ad\u20102597\t\r \u00a0(1996).\t\r \u00a094.\t\r \u00a0 M.\t\r \u00a0A.\t\r \u00a0Stein,\t\r \u00a0K.\t\r \u00a0Y.\t\r \u00a0Leung,\t\r \u00a0M.\t\r \u00a0Zwick,\t\r \u00a0F.\t\r \u00a0Garcia-\u00ad\u2010del\t\r \u00a0Portillo,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0Identification\t\r \u00a0of\t\r \u00a0a\t\r \u00a0Salmonella\t\r \u00a0virulence\t\r \u00a0gene\t\r \u00a0required\t\r \u00a0for\t\r \u00a0formation\t\r \u00a0of\t\r \u00a0filamentous\t\r \u00a0structures\t\r \u00a0containing\t\r \u00a0lysosomal\t\r \u00a0membrane\t\r \u00a0glycoproteins\t\r \u00a0within\t\r \u00a0epithelial\t\r \u00a0cells.\t\r \u00a0Mol\t\r \u00a0Microbiol\t\r \u00a020,\t\r \u00a0151-\u00ad\u2010164\t\r \u00a0(1996).\t\r \u00a095.\t\r \u00a0 P.\t\r \u00a0A.\t\r \u00a0Gulig,\t\r \u00a0H.\t\r \u00a0Danbara,\t\r \u00a0D.\t\r \u00a0G.\t\r \u00a0Guiney,\t\r \u00a0A.\t\r \u00a0J.\t\r \u00a0Lax,\t\r \u00a0F.\t\r \u00a0Norel,\t\r \u00a0M.\t\r \u00a0Rhen,\t\r \u00a0Molecular\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0spv\t\r \u00a0virulence\t\r \u00a0genes\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0virulence\t\r \u00a0plasmids.\t\r \u00a0Mol\t\r \u00a0 Microbiol\t\r \u00a07,\t\r \u00a0825-\u00ad\u2010830\t\r \u00a0(1993).\t\r \u00a096.\t\r \u00a0 S.\t\r \u00a0J.\t\r \u00a0Libby,\t\r \u00a0L.\t\r \u00a0G.\t\r \u00a0Adams,\t\r \u00a0T.\t\r \u00a0A.\t\r \u00a0Ficht,\t\r \u00a0C.\t\r \u00a0Allen,\t\r \u00a0H.\t\r \u00a0A.\t\r \u00a0Whitford,\t\r \u00a0N.\t\r \u00a0A.\t\r \u00a0Buchmeier,\t\r \u00a0S.\t\r \u00a0Bossie,\t\r \u00a0D.\t\r \u00a0G.\t\r \u00a0Guiney,\t\r \u00a0The\t\r \u00a0spv\t\r \u00a0genes\t\r \u00a0on\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0dublin\t\r \u00a0virulence\t\r \u00a0plasmid\t\r \u00a0are\t\r \u00a0required\t\r \u00a0for\t\r \u00a0severe\t\r \u00a0enteritis\t\r \u00a0and\t\r \u00a0systemic\t\r \u00a0infection\t\r \u00a0in\t\r \u00a0the\t\r \u00a0natural\t\r \u00a0host.\t\r \u00a0Infect\t\r \u00a0Immun\t\r \u00a065,\t\r \u00a01786-\u00ad\u20101792\t\r \u00a0(1997).\t\r \u00a097.\t\r \u00a0 R.\t\r \u00a0Huang,\t\r \u00a0S.\t\r \u00a0Wu,\t\r \u00a0X.\t\r \u00a0Zhang,\t\r \u00a0Y.\t\r \u00a0Zhang,\t\r \u00a0Molecular\t\r \u00a0analysis\t\r \u00a0and\t\r \u00a0identification\t\r \u00a0of\t\r \u00a0virulence\t\r \u00a0gene\t\r \u00a0on\t\r \u00a0pR(ST98)\t\r \u00a0from\t\r \u00a0multi-\u00ad\u2010drug\t\r \u00a0resistant\t\r \u00a0Salmonella\t\r \u00a0typhi.\t\r \u00a0Cell\t\r \u00a0 Mol\t\r \u00a0Immunol\t\r \u00a02,\t\r \u00a0136-\u00ad\u2010140\t\r \u00a0(2005).\t\r \u00a098.\t\r \u00a0 L.\t\r \u00a0R.\t\r \u00a0Prost,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0The\t\r \u00a0Salmonellae\t\r \u00a0PhoQ\t\r \u00a0sensor:\t\r \u00a0mechanisms\t\r \u00a0of\t\r \u00a0detection\t\r \u00a0of\t\r \u00a0phagosome\t\r \u00a0signals.\t\r \u00a0Cell\t\r \u00a0Microbiol\t\r \u00a010,\t\r \u00a0576-\u00ad\u2010582\t\r \u00a0(2008).\t\r \u00a099.\t\r \u00a0 I.\t\r \u00a0Behlau,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0A\t\r \u00a0PhoP-\u00ad\u2010repressed\t\r \u00a0gene\t\r \u00a0promotes\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0invasion\t\r \u00a0of\t\r \u00a0epithelial\t\r \u00a0cells.\t\r \u00a0J\t\r \u00a0Bacteriol\t\r \u00a0175,\t\r \u00a04475-\u00ad\u20104484\t\r \u00a0(1993).\t\r \u00a0100.\t\r \u00a0 L.\t\r \u00a0R.\t\r \u00a0Prost,\t\r \u00a0S.\t\r \u00a0Sanowar,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0Salmonella\t\r \u00a0sensing\t\r \u00a0of\t\r \u00a0anti-\u00ad\u2010microbial\t\r \u00a0mechanisms\t\r \u00a0to\t\r \u00a0promote\t\r \u00a0survival\t\r \u00a0within\t\r \u00a0macrophages.\t\r \u00a0Immunol\t\r \u00a0Rev\t\r \u00a0219,\t\r \u00a055-\u00ad\u201065\t\r \u00a0(2007).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 150\t\r \u00a0 101.\t\r \u00a0 C.\t\r \u00a0C.\t\r \u00a0Kim,\t\r \u00a0S.\t\r \u00a0Falkow,\t\r \u00a0Delineation\t\r \u00a0of\t\r \u00a0upstream\t\r \u00a0signaling\t\r \u00a0events\t\r \u00a0in\t\r \u00a0the\t\r \u00a0salmonella\t\r \u00a0pathogenicity\t\r \u00a0island\t\r \u00a02\t\r \u00a0transcriptional\t\r \u00a0activation\t\r \u00a0pathway.\t\r \u00a0J\t\r \u00a0 Bacteriol\t\r \u00a0186,\t\r \u00a04694-\u00ad\u20104704\t\r \u00a0(2004).\t\r \u00a0102.\t\r \u00a0 P.\t\r \u00a0I.\t\r \u00a0Fields,\t\r \u00a0E.\t\r \u00a0A.\t\r \u00a0Groisman,\t\r \u00a0F.\t\r \u00a0Heffron,\t\r \u00a0A\t\r \u00a0Salmonella\t\r \u00a0locus\t\r \u00a0that\t\r \u00a0controls\t\r \u00a0resistance\t\r \u00a0to\t\r \u00a0microbicidal\t\r \u00a0proteins\t\r \u00a0from\t\r \u00a0phagocytic\t\r \u00a0cells.\t\r \u00a0Science\t\r \u00a0243,\t\r \u00a01059-\u00ad\u20101062\t\r \u00a0(1989).\t\r \u00a0103.\t\r \u00a0 E.\t\r \u00a0A.\t\r \u00a0Groisman,\t\r \u00a0The\t\r \u00a0pleiotropic\t\r \u00a0two-\u00ad\u2010component\t\r \u00a0regulatory\t\r \u00a0system\t\r \u00a0PhoP-\u00ad\u2010PhoQ.\t\r \u00a0J\t\r \u00a0Bacteriol\t\r \u00a0183,\t\r \u00a01835-\u00ad\u20101842\t\r \u00a0(2001).\t\r \u00a0104.\t\r \u00a0 A.\t\r \u00a0J.\t\r \u00a0Baumler,\t\r \u00a0R.\t\r \u00a0M.\t\r \u00a0Tsolis,\t\r \u00a0T.\t\r \u00a0A.\t\r \u00a0Ficht,\t\r \u00a0L.\t\r \u00a0G.\t\r \u00a0Adams,\t\r \u00a0Evolution\t\r \u00a0of\t\r \u00a0host\t\r \u00a0adaptation\t\r \u00a0in\t\r \u00a0Salmonella\t\r \u00a0enterica.\t\r \u00a0Infect\t\r \u00a0Immun\t\r \u00a066,\t\r \u00a04579-\u00ad\u20104587\t\r \u00a0(1998).\t\r \u00a0105.\t\r \u00a0 S.\t\r \u00a0Porwollik,\t\r \u00a0R.\t\r \u00a0M.\t\r \u00a0Wong,\t\r \u00a0M.\t\r \u00a0McClelland,\t\r \u00a0Evolutionary\t\r \u00a0genomics\t\r \u00a0of\t\r \u00a0Salmonella:\t\r \u00a0gene\t\r \u00a0acquisitions\t\r \u00a0revealed\t\r \u00a0by\t\r \u00a0microarray\t\r \u00a0analysis.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0 Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a099,\t\r \u00a08956-\u00ad\u20108961\t\r \u00a0(2002).\t\r \u00a0106.\t\r \u00a0 T.\t\r \u00a0S.\t\r \u00a0Wallis,\t\r \u00a0E.\t\r \u00a0E.\t\r \u00a0Galyov,\t\r \u00a0Molecular\t\r \u00a0basis\t\r \u00a0of\t\r \u00a0Salmonella-\u00ad\u2010induced\t\r \u00a0enteritis.\t\r \u00a0Mol\t\r \u00a0 Microbiol\t\r \u00a036,\t\r \u00a0997-\u00ad\u20101005\t\r \u00a0(2000).\t\r \u00a0107.\t\r \u00a0 K.\t\r \u00a0H.\t\r \u00a0Darwin,\t\r \u00a0V.\t\r \u00a0L.\t\r \u00a0Miller,\t\r \u00a0Molecular\t\r \u00a0basis\t\r \u00a0of\t\r \u00a0the\t\r \u00a0interaction\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0with\t\r \u00a0the\t\r \u00a0intestinal\t\r \u00a0mucosa.\t\r \u00a0Clin\t\r \u00a0Microbiol\t\r \u00a0Rev\t\r \u00a012,\t\r \u00a0405-\u00ad\u2010428\t\r \u00a0(1999).\t\r \u00a0108.\t\r \u00a0 C.\t\r \u00a0Altier,\t\r \u00a0Genetic\t\r \u00a0and\t\r \u00a0environmental\t\r \u00a0control\t\r \u00a0of\t\r \u00a0salmonella\t\r \u00a0invasion.\t\r \u00a0J\t\r \u00a0 Microbiol\t\r \u00a043\t\r \u00a0Spec\t\r \u00a0No,\t\r \u00a085-\u00ad\u201092\t\r \u00a0(2005).\t\r \u00a0109.\t\r \u00a0 C.\t\r \u00a0P.\t\r \u00a0Lostroh,\t\r \u00a0C.\t\r \u00a0A.\t\r \u00a0Lee,\t\r \u00a0The\t\r \u00a0Salmonella\t\r \u00a0pathogenicity\t\r \u00a0island-\u00ad\u20101\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0system.\t\r \u00a0Microbes\t\r \u00a0Infect\t\r \u00a03,\t\r \u00a01281-\u00ad\u20101291\t\r \u00a0(2001).\t\r \u00a0110.\t\r \u00a0 C.\t\r \u00a0A.\t\r \u00a0Lee,\t\r \u00a0S.\t\r \u00a0Falkow,\t\r \u00a0The\t\r \u00a0ability\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0to\t\r \u00a0enter\t\r \u00a0mammalian\t\r \u00a0cells\t\r \u00a0is\t\r \u00a0affected\t\r \u00a0by\t\r \u00a0bacterial\t\r \u00a0growth\t\r \u00a0state.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a087,\t\r \u00a04304-\u00ad\u20104308\t\r \u00a0(1990).\t\r \u00a0111.\t\r \u00a0 U.\t\r \u00a0Lundberg,\t\r \u00a0U.\t\r \u00a0Vinatzer,\t\r \u00a0D.\t\r \u00a0Berdnik,\t\r \u00a0A.\t\r \u00a0von\t\r \u00a0Gabain,\t\r \u00a0M.\t\r \u00a0Baccarini,\t\r \u00a0Growth\t\r \u00a0phase-\u00ad\u2010regulated\t\r \u00a0induction\t\r \u00a0of\t\r \u00a0Salmonella-\u00ad\u2010induced\t\r \u00a0macrophage\t\r \u00a0apoptosis\t\r \u00a0correlates\t\r \u00a0with\t\r \u00a0transient\t\r \u00a0expression\t\r \u00a0of\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0genes.\t\r \u00a0J\t\r \u00a0Bacteriol\t\r \u00a0181,\t\r \u00a03433-\u00ad\u20103437\t\r \u00a0(1999).\t\r \u00a0112.\t\r \u00a0 R.\t\r \u00a0L.\t\r \u00a0Lucas,\t\r \u00a0C.\t\r \u00a0A.\t\r \u00a0Lee,\t\r \u00a0Roles\t\r \u00a0of\t\r \u00a0hilC\t\r \u00a0and\t\r \u00a0hilD\t\r \u00a0in\t\r \u00a0regulation\t\r \u00a0of\t\r \u00a0hilA\t\r \u00a0expression\t\r \u00a0in\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0Typhimurium.\t\r \u00a0J\t\r \u00a0Bacteriol\t\r \u00a0183,\t\r \u00a02733-\u00ad\u20102745\t\r \u00a0(2001).\t\r \u00a0113.\t\r \u00a0 C.\t\r \u00a0D.\t\r \u00a0Ellermeier,\t\r \u00a0J.\t\r \u00a0R.\t\r \u00a0Ellermeier,\t\r \u00a0J.\t\r \u00a0M.\t\r \u00a0Slauch,\t\r \u00a0HilD,\t\r \u00a0HilC\t\r \u00a0and\t\r \u00a0RtsA\t\r \u00a0constitute\t\r \u00a0a\t\r \u00a0feed\t\r \u00a0forward\t\r \u00a0loop\t\r \u00a0that\t\r \u00a0controls\t\r \u00a0expression\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SPI1\t\r \u00a0type\t\r \u00a0three\t\r \u00a0secretion\t\r \u00a0system\t\r \u00a0regulator\t\r \u00a0hilA\t\r \u00a0in\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0Typhimurium.\t\r \u00a0Mol\t\r \u00a0 Microbiol\t\r \u00a057,\t\r \u00a0691-\u00ad\u2010705\t\r \u00a0(2005).\t\r \u00a0114.\t\r \u00a0 J.\t\r \u00a0R.\t\r \u00a0Ellermeier,\t\r \u00a0J.\t\r \u00a0M.\t\r \u00a0Slauch,\t\r \u00a0Adaptation\t\r \u00a0to\t\r \u00a0the\t\r \u00a0host\t\r \u00a0environment:\t\r \u00a0regulation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0SPI1\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0system\t\r \u00a0in\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0Typhimurium.\t\r \u00a0Curr\t\r \u00a0Opin\t\r \u00a0Microbiol\t\r \u00a010,\t\r \u00a024-\u00ad\u201029\t\r \u00a0(2007).\t\r \u00a0115.\t\r \u00a0 M.\t\r \u00a0A.\t\r \u00a0Baxter,\t\r \u00a0T.\t\r \u00a0F.\t\r \u00a0Fahlen,\t\r \u00a0R.\t\r \u00a0L.\t\r \u00a0Wilson,\t\r \u00a0B.\t\r \u00a0D.\t\r \u00a0Jones,\t\r \u00a0HilE\t\r \u00a0interacts\t\r \u00a0with\t\r \u00a0HilD\t\r \u00a0and\t\r \u00a0negatively\t\r \u00a0regulates\t\r \u00a0hilA\t\r \u00a0transcription\t\r \u00a0and\t\r \u00a0expression\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0Typhimurium\t\r \u00a0invasive\t\r \u00a0phenotype.\t\r \u00a0Infect\t\r \u00a0Immun\t\r \u00a071,\t\r \u00a01295-\u00ad\u20101305\t\r \u00a0(2003).\t\r \u00a0116.\t\r \u00a0 M.\t\r \u00a0A.\t\r \u00a0Baxter,\t\r \u00a0B.\t\r \u00a0D.\t\r \u00a0Jones,\t\r \u00a0The\t\r \u00a0fimYZ\t\r \u00a0genes\t\r \u00a0regulate\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0Serovar\t\r \u00a0Typhimurium\t\r \u00a0invasion\t\r \u00a0in\t\r \u00a0addition\t\r \u00a0to\t\r \u00a0type\t\r \u00a01\t\r \u00a0fimbrial\t\r \u00a0expression\t\r \u00a0and\t\r \u00a0bacterial\t\r \u00a0motility.\t\r \u00a0Infect\t\r \u00a0Immun\t\r \u00a073,\t\r \u00a01377-\u00ad\u20101385\t\r \u00a0(2005).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 151\t\r \u00a0 117.\t\r \u00a0 B.\t\r \u00a0D.\t\r \u00a0Jones,\t\r \u00a0Salmonella\t\r \u00a0invasion\t\r \u00a0gene\t\r \u00a0regulation:\t\r \u00a0a\t\r \u00a0story\t\r \u00a0of\t\r \u00a0environmental\t\r \u00a0awareness.\t\r \u00a0J\t\r \u00a0Microbiol\t\r \u00a043\t\r \u00a0Spec\t\r \u00a0No,\t\r \u00a0110-\u00ad\u2010117\t\r \u00a0(2005).\t\r \u00a0118.\t\r \u00a0 J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0A.\t\r \u00a0Collmer,\t\r \u00a0Type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0machines:\t\r \u00a0bacterial\t\r \u00a0devices\t\r \u00a0for\t\r \u00a0protein\t\r \u00a0delivery\t\r \u00a0into\t\r \u00a0host\t\r \u00a0cells.\t\r \u00a0Science\t\r \u00a0284,\t\r \u00a01322-\u00ad\u20101328\t\r \u00a0(1999).\t\r \u00a0119.\t\r \u00a0 C.\t\r \u00a0J.\t\r \u00a0Hueck,\t\r \u00a0Type\t\r \u00a0III\t\r \u00a0protein\t\r \u00a0secretion\t\r \u00a0systems\t\r \u00a0in\t\r \u00a0bacterial\t\r \u00a0pathogens\t\r \u00a0of\t\r \u00a0animals\t\r \u00a0and\t\r \u00a0plants.\t\r \u00a0Microbiol\t\r \u00a0Mol\t\r \u00a0Biol\t\r \u00a0Rev\t\r \u00a062,\t\r \u00a0379-\u00ad\u2010433\t\r \u00a0(1998).\t\r \u00a0120.\t\r \u00a0 T.\t\r \u00a0Kubori,\t\r \u00a0Y.\t\r \u00a0Matsushima,\t\r \u00a0D.\t\r \u00a0Nakamura,\t\r \u00a0J.\t\r \u00a0Uralil,\t\r \u00a0M.\t\r \u00a0Lara-\u00ad\u2010Tejero,\t\r \u00a0A.\t\r \u00a0Sukhan,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Aizawa,\t\r \u00a0Supramolecular\t\r \u00a0structure\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0type\t\r \u00a0III\t\r \u00a0protein\t\r \u00a0secretion\t\r \u00a0system.\t\r \u00a0Science\t\r \u00a0280,\t\r \u00a0602-\u00ad\u2010605\t\r \u00a0(1998).\t\r \u00a0121.\t\r \u00a0 T.\t\r \u00a0Kubori,\t\r \u00a0A.\t\r \u00a0Sukhan,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Aizawa,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Molecular\t\r \u00a0characterization\t\r \u00a0and\t\r \u00a0assembly\t\r \u00a0of\t\r \u00a0the\t\r \u00a0needle\t\r \u00a0complex\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0type\t\r \u00a0III\t\r \u00a0protein\t\r \u00a0secretion\t\r \u00a0system.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a097,\t\r \u00a010225-\u00ad\u201010230\t\r \u00a0(2000).\t\r \u00a0122.\t\r \u00a0 T.\t\r \u00a0G.\t\r \u00a0Kimbrough,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0Contribution\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0components\t\r \u00a0to\t\r \u00a0needle\t\r \u00a0complex\t\r \u00a0formation.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a0 97,\t\r \u00a011008-\u00ad\u201011013\t\r \u00a0(2000).\t\r \u00a0123.\t\r \u00a0 A.\t\r \u00a0Sukhan,\t\r \u00a0T.\t\r \u00a0Kubori,\t\r \u00a0J.\t\r \u00a0Wilson,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Genetic\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0assembly\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0Typhimurium\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion-\u00ad\u2010associated\t\r \u00a0needle\t\r \u00a0complex.\t\r \u00a0J\t\r \u00a0Bacteriol\t\r \u00a0183,\t\r \u00a01159-\u00ad\u20101167\t\r \u00a0(2001).\t\r \u00a0124.\t\r \u00a0 T.\t\r \u00a0C.\t\r \u00a0Marlovits,\t\r \u00a0T.\t\r \u00a0Kubori,\t\r \u00a0A.\t\r \u00a0Sukhan,\t\r \u00a0D.\t\r \u00a0R.\t\r \u00a0Thomas,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0V.\t\r \u00a0M.\t\r \u00a0Unger,\t\r \u00a0Structural\t\r \u00a0insights\t\r \u00a0into\t\r \u00a0the\t\r \u00a0assembly\t\r \u00a0of\t\r \u00a0the\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0needle\t\r \u00a0complex.\t\r \u00a0 Science\t\r \u00a0306,\t\r \u00a01040-\u00ad\u20101042\t\r \u00a0(2004).\t\r \u00a0125.\t\r \u00a0 C.\t\r \u00a0K.\t\r \u00a0Yip,\t\r \u00a0T.\t\r \u00a0G.\t\r \u00a0Kimbrough,\t\r \u00a0H.\t\r \u00a0B.\t\r \u00a0Felise,\t\r \u00a0M.\t\r \u00a0Vuckovic,\t\r \u00a0N.\t\r \u00a0A.\t\r \u00a0Thomas,\t\r \u00a0R.\t\r \u00a0A.\t\r \u00a0Pfuetzner,\t\r \u00a0E.\t\r \u00a0A.\t\r \u00a0Frey,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0N.\t\r \u00a0C.\t\r \u00a0Strynadka,\t\r \u00a0Structural\t\r \u00a0characterization\t\r \u00a0of\t\r \u00a0the\t\r \u00a0molecular\t\r \u00a0platform\t\r \u00a0for\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0system\t\r \u00a0assembly.\t\r \u00a0Nature\t\r \u00a0435,\t\r \u00a0702-\u00ad\u2010707\t\r \u00a0(2005).\t\r \u00a0126.\t\r \u00a0 T.\t\r \u00a0C.\t\r \u00a0Marlovits,\t\r \u00a0T.\t\r \u00a0Kubori,\t\r \u00a0M.\t\r \u00a0Lara-\u00ad\u2010Tejero,\t\r \u00a0D.\t\r \u00a0Thomas,\t\r \u00a0V.\t\r \u00a0M.\t\r \u00a0Unger,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Assembly\t\r \u00a0of\t\r \u00a0the\t\r \u00a0inner\t\r \u00a0rod\t\r \u00a0determines\t\r \u00a0needle\t\r \u00a0length\t\r \u00a0in\t\r \u00a0the\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0injectisome.\t\r \u00a0Nature\t\r \u00a0441,\t\r \u00a0637-\u00ad\u2010640\t\r \u00a0(2006).\t\r \u00a0127.\t\r \u00a0 T.\t\r \u00a0Spreter,\t\r \u00a0C.\t\r \u00a0K.\t\r \u00a0Yip,\t\r \u00a0S.\t\r \u00a0Sanowar,\t\r \u00a0I.\t\r \u00a0Andre,\t\r \u00a0T.\t\r \u00a0G.\t\r \u00a0Kimbrough,\t\r \u00a0M.\t\r \u00a0Vuckovic,\t\r \u00a0R.\t\r \u00a0A.\t\r \u00a0Pfuetzner,\t\r \u00a0W.\t\r \u00a0Deng,\t\r \u00a0A.\t\r \u00a0C.\t\r \u00a0Yu,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0D.\t\r \u00a0Baker,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0N.\t\r \u00a0C.\t\r \u00a0Strynadka,\t\r \u00a0A\t\r \u00a0conserved\t\r \u00a0structural\t\r \u00a0motif\t\r \u00a0mediates\t\r \u00a0formation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0periplasmic\t\r \u00a0rings\t\r \u00a0in\t\r \u00a0the\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0system.\t\r \u00a0Nat\t\r \u00a0Struct\t\r \u00a0Mol\t\r \u00a0Biol\t\r \u00a016,\t\r \u00a0468-\u00ad\u2010476\t\r \u00a0(2009).\t\r \u00a0128.\t\r \u00a0 T.\t\r \u00a0G.\t\r \u00a0Kimbrough,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0Assembly\t\r \u00a0of\t\r \u00a0the\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0needle\t\r \u00a0complex\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0typhimurium.\t\r \u00a0Microbes\t\r \u00a0Infect\t\r \u00a04,\t\r \u00a075-\u00ad\u201082\t\r \u00a0(2002).\t\r \u00a0129.\t\r \u00a0 Y.\t\r \u00a0Akeda,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Genetic\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion-\u00ad\u2010associated\t\r \u00a0ATPase\t\r \u00a0InvC\t\r \u00a0defines\t\r \u00a0discrete\t\r \u00a0functional\t\r \u00a0domains.\t\r \u00a0J\t\r \u00a0 Bacteriol\t\r \u00a0186,\t\r \u00a02402-\u00ad\u20102412\t\r \u00a0(2004).\t\r \u00a0130.\t\r \u00a0 D.\t\r \u00a0Buttner,\t\r \u00a0U.\t\r \u00a0Bonas,\t\r \u00a0Port\t\r \u00a0of\t\r \u00a0entry-\u00ad\u2010-\u00ad\u2010the\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0translocon.\t\r \u00a0Trends\t\r \u00a0 Microbiol\t\r \u00a010,\t\r \u00a0186-\u00ad\u2010192\t\r \u00a0(2002).\t\r \u00a0131.\t\r \u00a0 K.\t\r \u00a0Kaniga,\t\r \u00a0D.\t\r \u00a0Trollinger,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Identification\t\r \u00a0of\t\r \u00a0two\t\r \u00a0targets\t\r \u00a0of\t\r \u00a0the\t\r \u00a0type\t\r \u00a0III\t\r \u00a0protein\t\r \u00a0secretion\t\r \u00a0system\t\r \u00a0encoded\t\r \u00a0by\t\r \u00a0the\t\r \u00a0inv\t\r \u00a0and\t\r \u00a0spa\t\r \u00a0loci\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0that\t\r \u00a0have\t\r \u00a0homology\t\r \u00a0to\t\r \u00a0the\t\r \u00a0Shigella\t\r \u00a0IpaD\t\r \u00a0and\t\r \u00a0IpaA\t\r \u00a0proteins.\t\r \u00a0J\t\r \u00a0 Bacteriol\t\r \u00a0177,\t\r \u00a07078-\u00ad\u20107085\t\r \u00a0(1995).\t\r \u00a0132.\t\r \u00a0 K.\t\r \u00a0Kaniga,\t\r \u00a0S.\t\r \u00a0Tucker,\t\r \u00a0D.\t\r \u00a0Trollinger,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Homologs\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Shigella\t\r \u00a0IpaB\t\r \u00a0and\t\r \u00a0IpaC\t\r \u00a0invasins\t\r \u00a0are\t\r \u00a0required\t\r \u00a0for\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0entry\t\r \u00a0into\t\r \u00a0cultured\t\r \u00a0epithelial\t\r \u00a0cells.\t\r \u00a0J\t\r \u00a0Bacteriol\t\r \u00a0177,\t\r \u00a03965-\u00ad\u20103971\t\r \u00a0(1995).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 152\t\r \u00a0 133.\t\r \u00a0 H.\t\r \u00a0G.\t\r \u00a0Kim,\t\r \u00a0B.\t\r \u00a0H.\t\r \u00a0Kim,\t\r \u00a0J.\t\r \u00a0S.\t\r \u00a0Kim,\t\r \u00a0J.\t\r \u00a0S.\t\r \u00a0Eom,\t\r \u00a0I.\t\r \u00a0S.\t\r \u00a0Bang,\t\r \u00a0S.\t\r \u00a0H.\t\r \u00a0Bang,\t\r \u00a0I.\t\r \u00a0S.\t\r \u00a0Lee,\t\r \u00a0Y.\t\r \u00a0K.\t\r \u00a0Park,\t\r \u00a0N-\u00ad\u2010terminal\t\r \u00a0residues\t\r \u00a0of\t\r \u00a0SipB\t\r \u00a0are\t\r \u00a0required\t\r \u00a0for\t\r \u00a0its\t\r \u00a0surface\t\r \u00a0localization\t\r \u00a0on\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0Typhimurium.\t\r \u00a0Microbiology\t\r \u00a0154,\t\r \u00a0207-\u00ad\u2010216\t\r \u00a0(2008).\t\r \u00a0134.\t\r \u00a0 R.\t\r \u00a0D.\t\r \u00a0Hayward,\t\r \u00a0E.\t\r \u00a0J.\t\r \u00a0McGhie,\t\r \u00a0V.\t\r \u00a0Koronakis,\t\r \u00a0Membrane\t\r \u00a0fusion\t\r \u00a0activity\t\r \u00a0of\t\r \u00a0purified\t\r \u00a0SipB,\t\r \u00a0a\t\r \u00a0Salmonella\t\r \u00a0surface\t\r \u00a0protein\t\r \u00a0essential\t\r \u00a0for\t\r \u00a0mammalian\t\r \u00a0cell\t\r \u00a0invasion.\t\r \u00a0Mol\t\r \u00a0Microbiol\t\r \u00a037,\t\r \u00a0727-\u00ad\u2010739\t\r \u00a0(2000).\t\r \u00a0135.\t\r \u00a0 M.\t\r \u00a0Lara-\u00ad\u2010Tejero,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0typhimurium\t\r \u00a0pathogenicity\t\r \u00a0island\t\r \u00a01-\u00ad\u2010encoded\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0system\t\r \u00a0translocases\t\r \u00a0mediate\t\r \u00a0intimate\t\r \u00a0attachment\t\r \u00a0to\t\r \u00a0nonphagocytic\t\r \u00a0cells.\t\r \u00a0Infect\t\r \u00a0Immun\t\r \u00a077,\t\r \u00a02635-\u00ad\u20102642\t\r \u00a0(2009).\t\r \u00a0136.\t\r \u00a0 C.\t\r \u00a0M.\t\r \u00a0Collazo,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0The\t\r \u00a0invasion-\u00ad\u2010associated\t\r \u00a0type\t\r \u00a0III\t\r \u00a0system\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0directs\t\r \u00a0the\t\r \u00a0translocation\t\r \u00a0of\t\r \u00a0Sip\t\r \u00a0proteins\t\r \u00a0into\t\r \u00a0the\t\r \u00a0host\t\r \u00a0cell.\t\r \u00a0Mol\t\r \u00a0Microbiol\t\r \u00a024,\t\r \u00a0747-\u00ad\u2010756\t\r \u00a0(1997).\t\r \u00a0137.\t\r \u00a0 C.\t\r \u00a0A.\t\r \u00a0Scherer,\t\r \u00a0E.\t\r \u00a0Cooper,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0The\t\r \u00a0Salmonella\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0translocon\t\r \u00a0protein\t\r \u00a0SspC\t\r \u00a0is\t\r \u00a0inserted\t\r \u00a0into\t\r \u00a0the\t\r \u00a0epithelial\t\r \u00a0cell\t\r \u00a0plasma\t\r \u00a0membrane\t\r \u00a0upon\t\r \u00a0infection.\t\r \u00a0Mol\t\r \u00a0Microbiol\t\r \u00a037,\t\r \u00a01133-\u00ad\u20101145\t\r \u00a0(2000).\t\r \u00a0138.\t\r \u00a0 R.\t\r \u00a0D.\t\r \u00a0Hayward,\t\r \u00a0R.\t\r \u00a0J.\t\r \u00a0Cain,\t\r \u00a0E.\t\r \u00a0J.\t\r \u00a0McGhie,\t\r \u00a0N.\t\r \u00a0Phillips,\t\r \u00a0M.\t\r \u00a0J.\t\r \u00a0Garner,\t\r \u00a0V.\t\r \u00a0Koronakis,\t\r \u00a0Cholesterol\t\r \u00a0binding\t\r \u00a0by\t\r \u00a0the\t\r \u00a0bacterial\t\r \u00a0type\t\r \u00a0III\t\r \u00a0translocon\t\r \u00a0is\t\r \u00a0essential\t\r \u00a0for\t\r \u00a0virulence\t\r \u00a0effector\t\r \u00a0delivery\t\r \u00a0into\t\r \u00a0mammalian\t\r \u00a0cells.\t\r \u00a0Mol\t\r \u00a0Microbiol\t\r \u00a056,\t\r \u00a0590-\u00ad\u2010603\t\r \u00a0(2005).\t\r \u00a0139.\t\r \u00a0 J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Common\t\r \u00a0themes\t\r \u00a0in\t\r \u00a0the\t\r \u00a0design\t\r \u00a0and\t\r \u00a0function\t\r \u00a0of\t\r \u00a0bacterial\t\r \u00a0effectors.\t\r \u00a0 Cell\t\r \u00a0Host\t\r \u00a0Microbe\t\r \u00a05,\t\r \u00a0571-\u00ad\u2010579\t\r \u00a0(2009).\t\r \u00a0140.\t\r \u00a0 E.\t\r \u00a0A.\t\r \u00a0Miao,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0A\t\r \u00a0conserved\t\r \u00a0amino\t\r \u00a0acid\t\r \u00a0sequence\t\r \u00a0directing\t\r \u00a0intracellular\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0by\t\r \u00a0Salmonella\t\r \u00a0typhimurium.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0 U\t\r \u00a0S\t\r \u00a0A\t\r \u00a097,\t\r \u00a07539-\u00ad\u20107544\t\r \u00a0(2000).\t\r \u00a0141.\t\r \u00a0 L.\t\r \u00a0S.\t\r \u00a0Collier-\u00ad\u2010Hyams,\t\r \u00a0H.\t\r \u00a0Zeng,\t\r \u00a0J.\t\r \u00a0Sun,\t\r \u00a0A.\t\r \u00a0D.\t\r \u00a0Tomlinson,\t\r \u00a0Z.\t\r \u00a0Q.\t\r \u00a0Bao,\t\r \u00a0H.\t\r \u00a0Chen,\t\r \u00a0J.\t\r \u00a0L.\t\r \u00a0Madara,\t\r \u00a0K.\t\r \u00a0Orth,\t\r \u00a0A.\t\r \u00a0S.\t\r \u00a0Neish,\t\r \u00a0Cutting\t\r \u00a0edge:\t\r \u00a0Salmonella\t\r \u00a0AvrA\t\r \u00a0effector\t\r \u00a0inhibits\t\r \u00a0the\t\r \u00a0key\t\r \u00a0proinflammatory,\t\r \u00a0anti-\u00ad\u2010apoptotic\t\r \u00a0NF-\u00ad\u2010kappa\t\r \u00a0B\t\r \u00a0pathway.\t\r \u00a0J\t\r \u00a0Immunol\t\r \u00a0 169,\t\r \u00a02846-\u00ad\u20102850\t\r \u00a0(2002).\t\r \u00a0142.\t\r \u00a0 Z.\t\r \u00a0Ye,\t\r \u00a0E.\t\r \u00a0O.\t\r \u00a0Petrof,\t\r \u00a0D.\t\r \u00a0Boone,\t\r \u00a0E.\t\r \u00a0C.\t\r \u00a0Claud,\t\r \u00a0J.\t\r \u00a0Sun,\t\r \u00a0Salmonella\t\r \u00a0effector\t\r \u00a0AvrA\t\r \u00a0regulation\t\r \u00a0of\t\r \u00a0colonic\t\r \u00a0epithelial\t\r \u00a0cell\t\r \u00a0inflammation\t\r \u00a0by\t\r \u00a0deubiquitination.\t\r \u00a0Am\t\r \u00a0J\t\r \u00a0 Pathol\t\r \u00a0171,\t\r \u00a0882-\u00ad\u2010892\t\r \u00a0(2007).\t\r \u00a0143.\t\r \u00a0 R.\t\r \u00a0M.\t\r \u00a0Jones,\t\r \u00a0H.\t\r \u00a0Wu,\t\r \u00a0C.\t\r \u00a0Wentworth,\t\r \u00a0L.\t\r \u00a0Luo,\t\r \u00a0L.\t\r \u00a0Collier-\u00ad\u2010Hyams,\t\r \u00a0A.\t\r \u00a0S.\t\r \u00a0Neish,\t\r \u00a0Salmonella\t\r \u00a0AvrA\t\r \u00a0Coordinates\t\r \u00a0Suppression\t\r \u00a0of\t\r \u00a0Host\t\r \u00a0Immune\t\r \u00a0and\t\r \u00a0Apoptotic\t\r \u00a0Defenses\t\r \u00a0via\t\r \u00a0JNK\t\r \u00a0Pathway\t\r \u00a0Blockade.\t\r \u00a0Cell\t\r \u00a0Host\t\r \u00a0Microbe\t\r \u00a03,\t\r \u00a0233-\u00ad\u2010244\t\r \u00a0(2008).\t\r \u00a0144.\t\r \u00a0 F.\t\r \u00a0Du,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Selective\t\r \u00a0inhibition\t\r \u00a0of\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0activated\t\r \u00a0signaling\t\r \u00a0by\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0effector\t\r \u00a0AvrA.\t\r \u00a0PLoS\t\r \u00a0Pathog\t\r \u00a05,\t\r \u00a0e1000595\t\r \u00a0(2009).\t\r \u00a0145.\t\r \u00a0 D.\t\r \u00a0Zhou,\t\r \u00a0M.\t\r \u00a0S.\t\r \u00a0Mooseker,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Role\t\r \u00a0of\t\r \u00a0the\t\r \u00a0S.\t\r \u00a0typhimurium\t\r \u00a0actin-\u00ad\u2010binding\t\r \u00a0protein\t\r \u00a0SipA\t\r \u00a0in\t\r \u00a0bacterial\t\r \u00a0internalization.\t\r \u00a0Science\t\r \u00a0283,\t\r \u00a02092-\u00ad\u20102095\t\r \u00a0(1999).\t\r \u00a0146.\t\r \u00a0 D.\t\r \u00a0Zhou,\t\r \u00a0M.\t\r \u00a0S.\t\r \u00a0Mooseker,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0An\t\r \u00a0invasion-\u00ad\u2010associated\t\r \u00a0Salmonella\t\r \u00a0protein\t\r \u00a0modulates\t\r \u00a0the\t\r \u00a0actin-\u00ad\u2010bundling\t\r \u00a0activity\t\r \u00a0of\t\r \u00a0plastin.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0 S\t\r \u00a0A\t\r \u00a096,\t\r \u00a010176-\u00ad\u201010181\t\r \u00a0(1999).\t\r \u00a0147.\t\r \u00a0 C.\t\r \u00a0A.\t\r \u00a0Lee,\t\r \u00a0M.\t\r \u00a0Silva,\t\r \u00a0A.\t\r \u00a0M.\t\r \u00a0Siber,\t\r \u00a0A.\t\r \u00a0J.\t\r \u00a0Kelly,\t\r \u00a0E.\t\r \u00a0Galyov,\t\r \u00a0B.\t\r \u00a0A.\t\r \u00a0McCormick,\t\r \u00a0A\t\r \u00a0secreted\t\r \u00a0Salmonella\t\r \u00a0protein\t\r \u00a0induces\t\r \u00a0a\t\r \u00a0proinflammatory\t\r \u00a0response\t\r \u00a0in\t\r \u00a0 \t\r \u00a0\t\r \u00a0 153\t\r \u00a0 epithelial\t\r \u00a0cells,\t\r \u00a0which\t\r \u00a0promotes\t\r \u00a0neutrophil\t\r \u00a0migration.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0 A\t\r \u00a097,\t\r \u00a012283-\u00ad\u201012288\t\r \u00a0(2000).\t\r \u00a0148.\t\r \u00a0 E.\t\r \u00a0C.\t\r \u00a0Boyle,\t\r \u00a0N.\t\r \u00a0F.\t\r \u00a0Brown,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0Typhimurium\t\r \u00a0effectors\t\r \u00a0SopB,\t\r \u00a0SopE,\t\r \u00a0SopE2\t\r \u00a0and\t\r \u00a0SipA\t\r \u00a0disrupt\t\r \u00a0tight\t\r \u00a0junction\t\r \u00a0structure\t\r \u00a0and\t\r \u00a0function.\t\r \u00a0Cell\t\r \u00a0Microbiol\t\r \u00a08,\t\r \u00a01946-\u00ad\u20101957\t\r \u00a0(2006).\t\r \u00a0149.\t\r \u00a0 L.\t\r \u00a0C.\t\r \u00a0Brawn,\t\r \u00a0R.\t\r \u00a0D.\t\r \u00a0Hayward,\t\r \u00a0V.\t\r \u00a0Koronakis,\t\r \u00a0Salmonella\t\r \u00a0SPI1\t\r \u00a0effector\t\r \u00a0SipA\t\r \u00a0persists\t\r \u00a0after\t\r \u00a0entry\t\r \u00a0and\t\r \u00a0cooperates\t\r \u00a0with\t\r \u00a0a\t\r \u00a0SPI2\t\r \u00a0effector\t\r \u00a0to\t\r \u00a0regulate\t\r \u00a0phagosome\t\r \u00a0maturation\t\r \u00a0and\t\r \u00a0intracellular\t\r \u00a0replication.\t\r \u00a0Cell\t\r \u00a0Host\t\r \u00a0Microbe\t\r \u00a01,\t\r \u00a063-\u00ad\u201075\t\r \u00a0(2007).\t\r \u00a0150.\t\r \u00a0 C.\t\r \u00a0V.\t\r \u00a0Srikanth,\t\r \u00a0D.\t\r \u00a0M.\t\r \u00a0Wall,\t\r \u00a0A.\t\r \u00a0Maldonado-\u00ad\u2010Contreras,\t\r \u00a0H.\t\r \u00a0N.\t\r \u00a0Shi,\t\r \u00a0D.\t\r \u00a0Zhou,\t\r \u00a0Z.\t\r \u00a0Demma,\t\r \u00a0K.\t\r \u00a0L.\t\r \u00a0Mumy,\t\r \u00a0B.\t\r \u00a0A.\t\r \u00a0McCormick,\t\r \u00a0Salmonella\t\r \u00a0pathogenesis\t\r \u00a0and\t\r \u00a0processing\t\r \u00a0of\t\r \u00a0secreted\t\r \u00a0effectors\t\r \u00a0by\t\r \u00a0caspase-\u00ad\u20103.\t\r \u00a0Science\t\r \u00a0330,\t\r \u00a0390-\u00ad\u2010393\t\r \u00a0(2010).\t\r \u00a0151.\t\r \u00a0 D.\t\r \u00a0Hersh,\t\r \u00a0D.\t\r \u00a0M.\t\r \u00a0Monack,\t\r \u00a0M.\t\r \u00a0R.\t\r \u00a0Smith,\t\r \u00a0N.\t\r \u00a0Ghori,\t\r \u00a0S.\t\r \u00a0Falkow,\t\r \u00a0A.\t\r \u00a0Zychlinsky,\t\r \u00a0The\t\r \u00a0Salmonella\t\r \u00a0invasin\t\r \u00a0SipB\t\r \u00a0induces\t\r \u00a0macrophage\t\r \u00a0apoptosis\t\r \u00a0by\t\r \u00a0binding\t\r \u00a0to\t\r \u00a0caspase-\u00ad\u20101.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a096,\t\r \u00a02396-\u00ad\u20102401\t\r \u00a0(1999).\t\r \u00a0152.\t\r \u00a0 V.\t\r \u00a0Jesenberger,\t\r \u00a0K.\t\r \u00a0J.\t\r \u00a0Procyk,\t\r \u00a0J.\t\r \u00a0Yuan,\t\r \u00a0S.\t\r \u00a0Reipert,\t\r \u00a0M.\t\r \u00a0Baccarini,\t\r \u00a0Salmonella-\u00ad\u2010induced\t\r \u00a0caspase-\u00ad\u20102\t\r \u00a0activation\t\r \u00a0in\t\r \u00a0macrophages:\t\r \u00a0a\t\r \u00a0novel\t\r \u00a0mechanism\t\r \u00a0in\t\r \u00a0pathogen-\u00ad\u2010mediated\t\r \u00a0apoptosis.\t\r \u00a0J\t\r \u00a0Exp\t\r \u00a0Med\t\r \u00a0192,\t\r \u00a01035-\u00ad\u20101046\t\r \u00a0(2000).\t\r \u00a0153.\t\r \u00a0 L.\t\r \u00a0D.\t\r \u00a0Hernandez,\t\r \u00a0M.\t\r \u00a0Pypaert,\t\r \u00a0R.\t\r \u00a0A.\t\r \u00a0Flavell,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0A\t\r \u00a0Salmonella\t\r \u00a0protein\t\r \u00a0causes\t\r \u00a0macrophage\t\r \u00a0cell\t\r \u00a0death\t\r \u00a0by\t\r \u00a0inducing\t\r \u00a0autophagy.\t\r \u00a0J\t\r \u00a0Cell\t\r \u00a0Biol\t\r \u00a0163,\t\r \u00a01123-\u00ad\u20101131\t\r \u00a0(2003).\t\r \u00a0154.\t\r \u00a0 R.\t\r \u00a0D.\t\r \u00a0Hayward,\t\r \u00a0V.\t\r \u00a0Koronakis,\t\r \u00a0Direct\t\r \u00a0nucleation\t\r \u00a0and\t\r \u00a0bundling\t\r \u00a0of\t\r \u00a0actin\t\r \u00a0by\t\r \u00a0the\t\r \u00a0SipC\t\r \u00a0protein\t\r \u00a0of\t\r \u00a0invasive\t\r \u00a0Salmonella.\t\r \u00a0EMBO\t\r \u00a0J\t\r \u00a018,\t\r \u00a04926-\u00ad\u20104934\t\r \u00a0(1999).\t\r \u00a0155.\t\r \u00a0 S.\t\r \u00a0K.\t\r \u00a0Myeni,\t\r \u00a0D.\t\r \u00a0Zhou,\t\r \u00a0The\t\r \u00a0C\t\r \u00a0terminus\t\r \u00a0of\t\r \u00a0SipC\t\r \u00a0binds\t\r \u00a0and\t\r \u00a0bundles\t\r \u00a0F-\u00ad\u2010actin\t\r \u00a0to\t\r \u00a0promote\t\r \u00a0Salmonella\t\r \u00a0invasion.\t\r \u00a0J\t\r \u00a0Biol\t\r \u00a0Chem\t\r \u00a0285,\t\r \u00a013357-\u00ad\u201013363\t\r \u00a0(2010).\t\r \u00a0156.\t\r \u00a0 J.\t\r \u00a0Chang,\t\r \u00a0J.\t\r \u00a0Chen,\t\r \u00a0D.\t\r \u00a0Zhou,\t\r \u00a0Delineation\t\r \u00a0and\t\r \u00a0characterization\t\r \u00a0of\t\r \u00a0the\t\r \u00a0actin\t\r \u00a0nucleation\t\r \u00a0and\t\r \u00a0effector\t\r \u00a0translocation\t\r \u00a0activities\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0SipC.\t\r \u00a0Mol\t\r \u00a0 Microbiol\t\r \u00a055,\t\r \u00a01379-\u00ad\u20101389\t\r \u00a0(2005).\t\r \u00a0157.\t\r \u00a0 J.\t\r \u00a0Chang,\t\r \u00a0S.\t\r \u00a0K.\t\r \u00a0Myeni,\t\r \u00a0T.\t\r \u00a0L.\t\r \u00a0Lin,\t\r \u00a0C.\t\r \u00a0C.\t\r \u00a0Wu,\t\r \u00a0C.\t\r \u00a0J.\t\r \u00a0Staiger,\t\r \u00a0D.\t\r \u00a0Zhou,\t\r \u00a0SipC\t\r \u00a0multimerization\t\r \u00a0promotes\t\r \u00a0actin\t\r \u00a0nucleation\t\r \u00a0and\t\r \u00a0contributes\t\r \u00a0to\t\r \u00a0Salmonella-\u00ad\u2010induced\t\r \u00a0inflammation.\t\r \u00a0Mol\t\r \u00a0Microbiol\t\r \u00a066,\t\r \u00a01548-\u00ad\u20101556\t\r \u00a0(2007).\t\r \u00a0158.\t\r \u00a0 E.\t\r \u00a0J.\t\r \u00a0McGhie,\t\r \u00a0R.\t\r \u00a0D.\t\r \u00a0Hayward,\t\r \u00a0V.\t\r \u00a0Koronakis,\t\r \u00a0Cooperation\t\r \u00a0between\t\r \u00a0actin-\u00ad\u2010binding\t\r \u00a0proteins\t\r \u00a0of\t\r \u00a0invasive\t\r \u00a0Salmonella:\t\r \u00a0SipA\t\r \u00a0potentiates\t\r \u00a0SipC\t\r \u00a0nucleation\t\r \u00a0and\t\r \u00a0bundling\t\r \u00a0of\t\r \u00a0actin.\t\r \u00a0EMBO\t\r \u00a0J\t\r \u00a020,\t\r \u00a02131-\u00ad\u20102139\t\r \u00a0(2001).\t\r \u00a0159.\t\r \u00a0 S.\t\r \u00a0A.\t\r \u00a0Carlson,\t\r \u00a0M.\t\r \u00a0B.\t\r \u00a0Omary,\t\r \u00a0B.\t\r \u00a0D.\t\r \u00a0Jones,\t\r \u00a0Identification\t\r \u00a0of\t\r \u00a0cytokeratins\t\r \u00a0as\t\r \u00a0accessory\t\r \u00a0mediators\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0entry\t\r \u00a0into\t\r \u00a0eukaryotic\t\r \u00a0cells.\t\r \u00a0Life\t\r \u00a0Sci\t\r \u00a070,\t\r \u00a01415-\u00ad\u20101426\t\r \u00a0(2002).\t\r \u00a0160.\t\r \u00a0 M.\t\r \u00a0Raffatellu,\t\r \u00a0R.\t\r \u00a0P.\t\r \u00a0Wilson,\t\r \u00a0D.\t\r \u00a0Chessa,\t\r \u00a0H.\t\r \u00a0Andrews-\u00ad\u2010Polymenis,\t\r \u00a0Q.\t\r \u00a0T.\t\r \u00a0Tran,\t\r \u00a0S.\t\r \u00a0Lawhon,\t\r \u00a0S.\t\r \u00a0Khare,\t\r \u00a0L.\t\r \u00a0G.\t\r \u00a0Adams,\t\r \u00a0A.\t\r \u00a0J.\t\r \u00a0Baumler,\t\r \u00a0SipA,\t\r \u00a0SopA,\t\r \u00a0SopB,\t\r \u00a0SopD,\t\r \u00a0and\t\r \u00a0SopE2\t\r \u00a0contribute\t\r \u00a0to\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serotype\t\r \u00a0typhimurium\t\r \u00a0invasion\t\r \u00a0of\t\r \u00a0epithelial\t\r \u00a0cells.\t\r \u00a0Infect\t\r \u00a0Immun\t\r \u00a073,\t\r \u00a0146-\u00ad\u2010154\t\r \u00a0(2005).\t\r \u00a0161.\t\r \u00a0 A.\t\r \u00a0N.\t\r \u00a0Layton,\t\r \u00a0P.\t\r \u00a0J.\t\r \u00a0Brown,\t\r \u00a0E.\t\r \u00a0E.\t\r \u00a0Galyov,\t\r \u00a0The\t\r \u00a0Salmonella\t\r \u00a0translocated\t\r \u00a0effector\t\r \u00a0SopA\t\r \u00a0is\t\r \u00a0targeted\t\r \u00a0to\t\r \u00a0the\t\r \u00a0mitochondria\t\r \u00a0of\t\r \u00a0infected\t\r \u00a0cells.\t\r \u00a0J\t\r \u00a0Bacteriol\t\r \u00a0187,\t\r \u00a03565-\u00ad\u20103571\t\r \u00a0(2005).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 154\t\r \u00a0 162.\t\r \u00a0 Y.\t\r \u00a0Zhang,\t\r \u00a0W.\t\r \u00a0M.\t\r \u00a0Higashide,\t\r \u00a0B.\t\r \u00a0A.\t\r \u00a0McCormick,\t\r \u00a0J.\t\r \u00a0Chen,\t\r \u00a0D.\t\r \u00a0Zhou,\t\r \u00a0The\t\r \u00a0inflammation-\u00ad\u2010associated\t\r \u00a0Salmonella\t\r \u00a0SopA\t\r \u00a0is\t\r \u00a0a\t\r \u00a0HECT-\u00ad\u2010like\t\r \u00a0E3\t\r \u00a0ubiquitin\t\r \u00a0ligase.\t\r \u00a0 Mol\t\r \u00a0Microbiol\t\r \u00a062,\t\r \u00a0786-\u00ad\u2010793\t\r \u00a0(2006).\t\r \u00a0163.\t\r \u00a0 S.\t\r \u00a0L.\t\r \u00a0Marcus,\t\r \u00a0M.\t\r \u00a0R.\t\r \u00a0Wenk,\t\r \u00a0O.\t\r \u00a0Steele-\u00ad\u2010Mortimer,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0A\t\r \u00a0synaptojanin-\u00ad\u2010homologous\t\r \u00a0region\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0SigD\t\r \u00a0is\t\r \u00a0essential\t\r \u00a0for\t\r \u00a0inositol\t\r \u00a0phosphatase\t\r \u00a0activity\t\r \u00a0and\t\r \u00a0Akt\t\r \u00a0activation.\t\r \u00a0FEBS\t\r \u00a0Lett\t\r \u00a0494,\t\r \u00a0201-\u00ad\u2010207\t\r \u00a0(2001).\t\r \u00a0164.\t\r \u00a0 L.\t\r \u00a0S.\t\r \u00a0Bertelsen,\t\r \u00a0G.\t\r \u00a0Paesold,\t\r \u00a0S.\t\r \u00a0L.\t\r \u00a0Marcus,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0L.\t\r \u00a0Eckmann,\t\r \u00a0K.\t\r \u00a0E.\t\r \u00a0Barrett,\t\r \u00a0Modulation\t\r \u00a0of\t\r \u00a0chloride\t\r \u00a0secretory\t\r \u00a0responses\t\r \u00a0and\t\r \u00a0barrier\t\r \u00a0function\t\r \u00a0of\t\r \u00a0intestinal\t\r \u00a0epithelial\t\r \u00a0cells\t\r \u00a0by\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0effector\t\r \u00a0protein\t\r \u00a0SigD.\t\r \u00a0Am\t\r \u00a0J\t\r \u00a0Physiol\t\r \u00a0Cell\t\r \u00a0 Physiol\t\r \u00a0287,\t\r \u00a0C939-\u00ad\u2010948\t\r \u00a0(2004).\t\r \u00a0165.\t\r \u00a0 L.\t\r \u00a0A.\t\r \u00a0Knodler,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0O.\t\r \u00a0Steele-\u00ad\u2010Mortimer,\t\r \u00a0The\t\r \u00a0Salmonella\t\r \u00a0effector\t\r \u00a0protein\t\r \u00a0SopB\t\r \u00a0protects\t\r \u00a0epithelial\t\r \u00a0cells\t\r \u00a0from\t\r \u00a0apoptosis\t\r \u00a0by\t\r \u00a0sustained\t\r \u00a0activation\t\r \u00a0of\t\r \u00a0Akt.\t\r \u00a0J\t\r \u00a0 Biol\t\r \u00a0Chem\t\r \u00a0280,\t\r \u00a09058-\u00ad\u20109064\t\r \u00a0(2005).\t\r \u00a0166.\t\r \u00a0 L.\t\r \u00a0D.\t\r \u00a0Rogers,\t\r \u00a0A.\t\r \u00a0R.\t\r \u00a0Kristensen,\t\r \u00a0E.\t\r \u00a0C.\t\r \u00a0Boyle,\t\r \u00a0D.\t\r \u00a0P.\t\r \u00a0Robinson,\t\r \u00a0R.\t\r \u00a0T.\t\r \u00a0Ly,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0L.\t\r \u00a0J.\t\r \u00a0Foster,\t\r \u00a0Identification\t\r \u00a0of\t\r \u00a0cognate\t\r \u00a0host\t\r \u00a0targets\t\r \u00a0and\t\r \u00a0specific\t\r \u00a0ubiquitylation\t\r \u00a0sites\t\r \u00a0on\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0effector\t\r \u00a0SopB\/SigD.\t\r \u00a0J\t\r \u00a0Proteomics\t\r \u00a071,\t\r \u00a097-\u00ad\u2010108\t\r \u00a0(2008).\t\r \u00a0167.\t\r \u00a0 M.\t\r \u00a0A.\t\r \u00a0Bakowski,\t\r \u00a0J.\t\r \u00a0T.\t\r \u00a0Cirulis,\t\r \u00a0N.\t\r \u00a0F.\t\r \u00a0Brown,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0J.\t\r \u00a0H.\t\r \u00a0Brumell,\t\r \u00a0SopD\t\r \u00a0acts\t\r \u00a0cooperatively\t\r \u00a0with\t\r \u00a0SopB\t\r \u00a0during\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0Typhimurium\t\r \u00a0invasion.\t\r \u00a0Cell\t\r \u00a0Microbiol\t\r \u00a09,\t\r \u00a02839-\u00ad\u20102855\t\r \u00a0(2007).\t\r \u00a0168.\t\r \u00a0 W.\t\r \u00a0D.\t\r \u00a0Hardt,\t\r \u00a0L.\t\r \u00a0M.\t\r \u00a0Chen,\t\r \u00a0K.\t\r \u00a0E.\t\r \u00a0Schuebel,\t\r \u00a0X.\t\r \u00a0R.\t\r \u00a0Bustelo,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0S.\t\r \u00a0typhimurium\t\r \u00a0encodes\t\r \u00a0an\t\r \u00a0activator\t\r \u00a0of\t\r \u00a0Rho\t\r \u00a0GTPases\t\r \u00a0that\t\r \u00a0induces\t\r \u00a0membrane\t\r \u00a0ruffling\t\r \u00a0and\t\r \u00a0nuclear\t\r \u00a0responses\t\r \u00a0in\t\r \u00a0host\t\r \u00a0cells.\t\r \u00a0Cell\t\r \u00a093,\t\r \u00a0815-\u00ad\u2010826\t\r \u00a0(1998).\t\r \u00a0169.\t\r \u00a0 M.\t\r \u00a0G.\t\r \u00a0Rudolph,\t\r \u00a0C.\t\r \u00a0Weise,\t\r \u00a0S.\t\r \u00a0Mirold,\t\r \u00a0B.\t\r \u00a0Hillenbrand,\t\r \u00a0B.\t\r \u00a0Bader,\t\r \u00a0A.\t\r \u00a0Wittinghofer,\t\r \u00a0W.\t\r \u00a0D.\t\r \u00a0Hardt,\t\r \u00a0Biochemical\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0SopE\t\r \u00a0from\t\r \u00a0Salmonella\t\r \u00a0typhimurium,\t\r \u00a0a\t\r \u00a0highly\t\r \u00a0efficient\t\r \u00a0guanosine\t\r \u00a0nucleotide\t\r \u00a0exchange\t\r \u00a0factor\t\r \u00a0for\t\r \u00a0RhoGTPases.\t\r \u00a0J\t\r \u00a0Biol\t\r \u00a0 Chem\t\r \u00a0274,\t\r \u00a030501-\u00ad\u201030509\t\r \u00a0(1999).\t\r \u00a0170.\t\r \u00a0 K.\t\r \u00a0Mukherjee,\t\r \u00a0S.\t\r \u00a0Parashuraman,\t\r \u00a0M.\t\r \u00a0Raje,\t\r \u00a0A.\t\r \u00a0Mukhopadhyay,\t\r \u00a0SopE\t\r \u00a0acts\t\r \u00a0as\t\r \u00a0an\t\r \u00a0Rab5-\u00ad\u2010specific\t\r \u00a0nucleotide\t\r \u00a0exchange\t\r \u00a0factor\t\r \u00a0and\t\r \u00a0recruits\t\r \u00a0non-\u00ad\u2010prenylated\t\r \u00a0Rab5\t\r \u00a0on\t\r \u00a0Salmonella-\u00ad\u2010containing\t\r \u00a0phagosomes\t\r \u00a0to\t\r \u00a0promote\t\r \u00a0fusion\t\r \u00a0with\t\r \u00a0early\t\r \u00a0endosomes.\t\r \u00a0J\t\r \u00a0Biol\t\r \u00a0Chem\t\r \u00a0276,\t\r \u00a023607-\u00ad\u201023615\t\r \u00a0(2001).\t\r \u00a0171.\t\r \u00a0 R.\t\r \u00a0Madan,\t\r \u00a0G.\t\r \u00a0Krishnamurthy,\t\r \u00a0A.\t\r \u00a0Mukhopadhyay,\t\r \u00a0SopE-\u00ad\u2010mediated\t\r \u00a0recruitment\t\r \u00a0of\t\r \u00a0host\t\r \u00a0Rab5\t\r \u00a0on\t\r \u00a0phagosomes\t\r \u00a0inhibits\t\r \u00a0Salmonella\t\r \u00a0transport\t\r \u00a0to\t\r \u00a0lysosomes.\t\r \u00a0 Methods\t\r \u00a0Mol\t\r \u00a0Biol\t\r \u00a0445,\t\r \u00a0417-\u00ad\u2010437\t\r \u00a0(2008).\t\r \u00a0172.\t\r \u00a0 A.\t\r \u00a0J.\t\r \u00a0Muller,\t\r \u00a0C.\t\r \u00a0Hoffmann,\t\r \u00a0M.\t\r \u00a0Galle,\t\r \u00a0A.\t\r \u00a0Van\t\r \u00a0Den\t\r \u00a0Broeke,\t\r \u00a0M.\t\r \u00a0Heikenwalder,\t\r \u00a0L.\t\r \u00a0Falter,\t\r \u00a0B.\t\r \u00a0Misselwitz,\t\r \u00a0M.\t\r \u00a0Kremer,\t\r \u00a0R.\t\r \u00a0Beyaert,\t\r \u00a0W.\t\r \u00a0D.\t\r \u00a0Hardt,\t\r \u00a0The\t\r \u00a0S.\t\r \u00a0Typhimurium\t\r \u00a0effector\t\r \u00a0SopE\t\r \u00a0induces\t\r \u00a0caspase-\u00ad\u20101\t\r \u00a0activation\t\r \u00a0in\t\r \u00a0stromal\t\r \u00a0cells\t\r \u00a0to\t\r \u00a0initiate\t\r \u00a0gut\t\r \u00a0inflammation.\t\r \u00a0Cell\t\r \u00a0Host\t\r \u00a0Microbe\t\r \u00a06,\t\r \u00a0125-\u00ad\u2010136\t\r \u00a0(2009).\t\r \u00a0173.\t\r \u00a0 S.\t\r \u00a0Stender,\t\r \u00a0A.\t\r \u00a0Friebel,\t\r \u00a0S.\t\r \u00a0Linder,\t\r \u00a0M.\t\r \u00a0Rohde,\t\r \u00a0S.\t\r \u00a0Mirold,\t\r \u00a0W.\t\r \u00a0D.\t\r \u00a0Hardt,\t\r \u00a0Identification\t\r \u00a0of\t\r \u00a0SopE2\t\r \u00a0from\t\r \u00a0Salmonella\t\r \u00a0typhimurium,\t\r \u00a0a\t\r \u00a0conserved\t\r \u00a0guanine\t\r \u00a0nucleotide\t\r \u00a0exchange\t\r \u00a0factor\t\r \u00a0for\t\r \u00a0Cdc42\t\r \u00a0of\t\r \u00a0the\t\r \u00a0host\t\r \u00a0cell.\t\r \u00a0Mol\t\r \u00a0Microbiol\t\r \u00a036,\t\r \u00a01206-\u00ad\u20101221\t\r \u00a0(2000).\t\r \u00a0174.\t\r \u00a0 A.\t\r \u00a0Friebel,\t\r \u00a0H.\t\r \u00a0Ilchmann,\t\r \u00a0M.\t\r \u00a0Aepfelbacher,\t\r \u00a0K.\t\r \u00a0Ehrbar,\t\r \u00a0W.\t\r \u00a0Machleidt,\t\r \u00a0W.\t\r \u00a0D.\t\r \u00a0Hardt,\t\r \u00a0SopE\t\r \u00a0and\t\r \u00a0SopE2\t\r \u00a0from\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0activate\t\r \u00a0different\t\r \u00a0sets\t\r \u00a0of\t\r \u00a0RhoGTPases\t\r \u00a0of\t\r \u00a0the\t\r \u00a0host\t\r \u00a0cell.\t\r \u00a0J\t\r \u00a0Biol\t\r \u00a0Chem\t\r \u00a0276,\t\r \u00a034035-\u00ad\u201034040\t\r \u00a0(2001).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 155\t\r \u00a0 175.\t\r \u00a0 K.\t\r \u00a0Kaniga,\t\r \u00a0J.\t\r \u00a0Uralil,\t\r \u00a0J.\t\r \u00a0B.\t\r \u00a0Bliska,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0A\t\r \u00a0secreted\t\r \u00a0protein\t\r \u00a0tyrosine\t\r \u00a0phosphatase\t\r \u00a0with\t\r \u00a0modular\t\r \u00a0effector\t\r \u00a0domains\t\r \u00a0in\t\r \u00a0the\t\r \u00a0bacterial\t\r \u00a0pathogen\t\r \u00a0Salmonella\t\r \u00a0typhimurium.\t\r \u00a0Mol\t\r \u00a0Microbiol\t\r \u00a021,\t\r \u00a0633-\u00ad\u2010641\t\r \u00a0(1996).\t\r \u00a0176.\t\r \u00a0 Y.\t\r \u00a0Fu,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0A\t\r \u00a0salmonella\t\r \u00a0protein\t\r \u00a0antagonizes\t\r \u00a0Rac-\u00ad\u20101\t\r \u00a0and\t\r \u00a0Cdc42\t\r \u00a0to\t\r \u00a0mediate\t\r \u00a0host-\u00ad\u2010cell\t\r \u00a0recovery\t\r \u00a0after\t\r \u00a0bacterial\t\r \u00a0invasion.\t\r \u00a0Nature\t\r \u00a0401,\t\r \u00a0293-\u00ad\u2010297\t\r \u00a0(1999).\t\r \u00a0177.\t\r \u00a0 S.\t\r \u00a0Murli,\t\r \u00a0R.\t\r \u00a0O.\t\r \u00a0Watson,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Role\t\r \u00a0of\t\r \u00a0tyrosine\t\r \u00a0kinases\t\r \u00a0and\t\r \u00a0the\t\r \u00a0tyrosine\t\r \u00a0phosphatase\t\r \u00a0SptP\t\r \u00a0in\t\r \u00a0the\t\r \u00a0interaction\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0with\t\r \u00a0host\t\r \u00a0cells.\t\r \u00a0Cell\t\r \u00a0 Microbiol\t\r \u00a03,\t\r \u00a0795-\u00ad\u2010810\t\r \u00a0(2001).\t\r \u00a0178.\t\r \u00a0 S.\t\r \u00a0L.\t\r \u00a0Lin,\t\r \u00a0T.\t\r \u00a0X.\t\r \u00a0Le,\t\r \u00a0D.\t\r \u00a0S.\t\r \u00a0Cowen,\t\r \u00a0SptP,\t\r \u00a0a\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0type\t\r \u00a0III-\u00ad\u2010secreted\t\r \u00a0protein,\t\r \u00a0inhibits\t\r \u00a0the\t\r \u00a0mitogen-\u00ad\u2010activated\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a0pathway\t\r \u00a0by\t\r \u00a0inhibiting\t\r \u00a0Raf\t\r \u00a0activation.\t\r \u00a0Cell\t\r \u00a0Microbiol\t\r \u00a05,\t\r \u00a0267-\u00ad\u2010275\t\r \u00a0(2003).\t\r \u00a0179.\t\r \u00a0 D.\t\r \u00a0Humphreys,\t\r \u00a0P.\t\r \u00a0J.\t\r \u00a0Hume,\t\r \u00a0V.\t\r \u00a0Koronakis,\t\r \u00a0The\t\r \u00a0Salmonella\t\r \u00a0effector\t\r \u00a0SptP\t\r \u00a0dephosphorylates\t\r \u00a0host\t\r \u00a0AAA+\t\r \u00a0ATPase\t\r \u00a0VCP\t\r \u00a0to\t\r \u00a0promote\t\r \u00a0development\t\r \u00a0of\t\r \u00a0its\t\r \u00a0intracellular\t\r \u00a0replicative\t\r \u00a0niche.\t\r \u00a0Cell\t\r \u00a0Host\t\r \u00a0Microbe\t\r \u00a05,\t\r \u00a0225-\u00ad\u2010233\t\r \u00a0(2009).\t\r \u00a0180.\t\r \u00a0 J.\t\r \u00a0Bernal-\u00ad\u2010Bayard,\t\r \u00a0F.\t\r \u00a0Ramos-\u00ad\u2010Morales,\t\r \u00a0Salmonella\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0effector\t\r \u00a0SlrP\t\r \u00a0is\t\r \u00a0an\t\r \u00a0E3\t\r \u00a0ubiquitin\t\r \u00a0ligase\t\r \u00a0for\t\r \u00a0mammalian\t\r \u00a0thioredoxin.\t\r \u00a0J\t\r \u00a0Biol\t\r \u00a0Chem\t\r \u00a0284,\t\r \u00a027587-\u00ad\u201027595\t\r \u00a0(2009).\t\r \u00a0181.\t\r \u00a0 J.\t\r \u00a0Bernal-\u00ad\u2010Bayard,\t\r \u00a0E.\t\r \u00a0Cardenal-\u00ad\u2010Munoz,\t\r \u00a0F.\t\r \u00a0Ramos-\u00ad\u2010Morales,\t\r \u00a0The\t\r \u00a0Salmonella\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0effector,\t\r \u00a0salmonella\t\r \u00a0leucine-\u00ad\u2010rich\t\r \u00a0repeat\t\r \u00a0protein\t\r \u00a0(SlrP),\t\r \u00a0targets\t\r \u00a0the\t\r \u00a0human\t\r \u00a0chaperone\t\r \u00a0ERdj3.\t\r \u00a0J\t\r \u00a0Biol\t\r \u00a0Chem\t\r \u00a0285,\t\r \u00a016360-\u00ad\u201016368\t\r \u00a0(2010).\t\r \u00a0182.\t\r \u00a0 A.\t\r \u00a0Haraga,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0A\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0typhimurium\t\r \u00a0translocated\t\r \u00a0leucine-\u00ad\u2010rich\t\r \u00a0repeat\t\r \u00a0effector\t\r \u00a0protein\t\r \u00a0inhibits\t\r \u00a0NF-\u00ad\u2010kappa\t\r \u00a0B-\u00ad\u2010dependent\t\r \u00a0gene\t\r \u00a0expression.\t\r \u00a0Infect\t\r \u00a0Immun\t\r \u00a071,\t\r \u00a04052-\u00ad\u20104058\t\r \u00a0(2003).\t\r \u00a0183.\t\r \u00a0 A.\t\r \u00a0Haraga,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0A\t\r \u00a0Salmonella\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0effector\t\r \u00a0interacts\t\r \u00a0with\t\r \u00a0the\t\r \u00a0mammalian\t\r \u00a0serine\/threonine\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a0PKN1.\t\r \u00a0Cell\t\r \u00a0Microbiol\t\r \u00a08,\t\r \u00a0837-\u00ad\u2010846\t\r \u00a0(2006).\t\r \u00a0184.\t\r \u00a0 J.\t\r \u00a0R.\t\r \u00a0Rohde,\t\r \u00a0A.\t\r \u00a0Breitkreutz,\t\r \u00a0A.\t\r \u00a0Chenal,\t\r \u00a0P.\t\r \u00a0J.\t\r \u00a0Sansonetti,\t\r \u00a0C.\t\r \u00a0Parsot,\t\r \u00a0Type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0effectors\t\r \u00a0of\t\r \u00a0the\t\r \u00a0IpaH\t\r \u00a0family\t\r \u00a0are\t\r \u00a0E3\t\r \u00a0ubiquitin\t\r \u00a0ligases.\t\r \u00a0Cell\t\r \u00a0Host\t\r \u00a0 Microbe\t\r \u00a01,\t\r \u00a077-\u00ad\u201083\t\r \u00a0(2007).\t\r \u00a0185.\t\r \u00a0 F.\t\r \u00a0A.\t\r \u00a0Norris,\t\r \u00a0M.\t\r \u00a0P.\t\r \u00a0Wilson,\t\r \u00a0T.\t\r \u00a0S.\t\r \u00a0Wallis,\t\r \u00a0E.\t\r \u00a0E.\t\r \u00a0Galyov,\t\r \u00a0P.\t\r \u00a0W.\t\r \u00a0Majerus,\t\r \u00a0SopB,\t\r \u00a0a\t\r \u00a0protein\t\r \u00a0required\t\r \u00a0for\t\r \u00a0virulence\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0dublin,\t\r \u00a0is\t\r \u00a0an\t\r \u00a0inositol\t\r \u00a0phosphate\t\r \u00a0phosphatase.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a095,\t\r \u00a014057-\u00ad\u201014059\t\r \u00a0(1998).\t\r \u00a0186.\t\r \u00a0 C.\t\r \u00a0S.\t\r \u00a0Bakshi,\t\r \u00a0V.\t\r \u00a0P.\t\r \u00a0Singh,\t\r \u00a0M.\t\r \u00a0W.\t\r \u00a0Wood,\t\r \u00a0P.\t\r \u00a0W.\t\r \u00a0Jones,\t\r \u00a0T.\t\r \u00a0S.\t\r \u00a0Wallis,\t\r \u00a0E.\t\r \u00a0E.\t\r \u00a0Galyov,\t\r \u00a0Identification\t\r \u00a0of\t\r \u00a0SopE2,\t\r \u00a0a\t\r \u00a0Salmonella\t\r \u00a0secreted\t\r \u00a0protein\t\r \u00a0which\t\r \u00a0is\t\r \u00a0highly\t\r \u00a0homologous\t\r \u00a0to\t\r \u00a0SopE\t\r \u00a0and\t\r \u00a0involved\t\r \u00a0in\t\r \u00a0bacterial\t\r \u00a0invasion\t\r \u00a0of\t\r \u00a0epithelial\t\r \u00a0cells.\t\r \u00a0J\t\r \u00a0 Bacteriol\t\r \u00a0182,\t\r \u00a02341-\u00ad\u20102344\t\r \u00a0(2000).\t\r \u00a0187.\t\r \u00a0 D.\t\r \u00a0Zhou,\t\r \u00a0L.\t\r \u00a0M.\t\r \u00a0Chen,\t\r \u00a0L.\t\r \u00a0Hernandez,\t\r \u00a0S.\t\r \u00a0B.\t\r \u00a0Shears,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0A\t\r \u00a0Salmonella\t\r \u00a0inositol\t\r \u00a0polyphosphatase\t\r \u00a0acts\t\r \u00a0in\t\r \u00a0conjunction\t\r \u00a0with\t\r \u00a0other\t\r \u00a0bacterial\t\r \u00a0effectors\t\r \u00a0to\t\r \u00a0promote\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0actin\t\r \u00a0cytoskeleton\t\r \u00a0rearrangements\t\r \u00a0and\t\r \u00a0bacterial\t\r \u00a0internalization.\t\r \u00a0Mol\t\r \u00a0Microbiol\t\r \u00a039,\t\r \u00a0248-\u00ad\u2010259\t\r \u00a0(2001).\t\r \u00a0188.\t\r \u00a0 W.\t\r \u00a0Higashide,\t\r \u00a0S.\t\r \u00a0Dai,\t\r \u00a0V.\t\r \u00a0P.\t\r \u00a0Hombs,\t\r \u00a0D.\t\r \u00a0Zhou,\t\r \u00a0Involvement\t\r \u00a0of\t\r \u00a0SipA\t\r \u00a0in\t\r \u00a0modulating\t\r \u00a0actin\t\r \u00a0dynamics\t\r \u00a0during\t\r \u00a0Salmonella\t\r \u00a0invasion\t\r \u00a0into\t\r \u00a0cultured\t\r \u00a0epithelial\t\r \u00a0cells.\t\r \u00a0Cell\t\r \u00a0 Microbiol\t\r \u00a04,\t\r \u00a0357-\u00ad\u2010365\t\r \u00a0(2002).\t\r \u00a0189.\t\r \u00a0 V.\t\r \u00a0M.\t\r \u00a0Bruno,\t\r \u00a0S.\t\r \u00a0Hannemann,\t\r \u00a0M.\t\r \u00a0Lara-\u00ad\u2010Tejero,\t\r \u00a0R.\t\r \u00a0A.\t\r \u00a0Flavell,\t\r \u00a0S.\t\r \u00a0H.\t\r \u00a0Kleinstein,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Salmonella\t\r \u00a0Typhimurium\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0effectors\t\r \u00a0stimulate\t\r \u00a0innate\t\r \u00a0 \t\r \u00a0\t\r \u00a0 156\t\r \u00a0 immune\t\r \u00a0responses\t\r \u00a0in\t\r \u00a0cultured\t\r \u00a0epithelial\t\r \u00a0cells.\t\r \u00a0PLoS\t\r \u00a0Pathog\t\r \u00a05,\t\r \u00a0e1000538\t\r \u00a0(2009).\t\r \u00a0190.\t\r \u00a0 B.\t\r \u00a0Stecher,\t\r \u00a0R.\t\r \u00a0Robbiani,\t\r \u00a0A.\t\r \u00a0W.\t\r \u00a0Walker,\t\r \u00a0A.\t\r \u00a0M.\t\r \u00a0Westendorf,\t\r \u00a0M.\t\r \u00a0Barthel,\t\r \u00a0M.\t\r \u00a0Kremer,\t\r \u00a0S.\t\r \u00a0Chaffron,\t\r \u00a0A.\t\r \u00a0J.\t\r \u00a0Macpherson,\t\r \u00a0J.\t\r \u00a0Buer,\t\r \u00a0J.\t\r \u00a0Parkhill,\t\r \u00a0G.\t\r \u00a0Dougan,\t\r \u00a0C.\t\r \u00a0von\t\r \u00a0Mering,\t\r \u00a0W.\t\r \u00a0D.\t\r \u00a0Hardt,\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0typhimurium\t\r \u00a0exploits\t\r \u00a0inflammation\t\r \u00a0to\t\r \u00a0compete\t\r \u00a0with\t\r \u00a0the\t\r \u00a0intestinal\t\r \u00a0microbiota.\t\r \u00a0PLoS\t\r \u00a0Biol\t\r \u00a05,\t\r \u00a02177-\u00ad\u20102189\t\r \u00a0(2007).\t\r \u00a0191.\t\r \u00a0 S.\t\r \u00a0E.\t\r \u00a0Winter,\t\r \u00a0P.\t\r \u00a0Thiennimitr,\t\r \u00a0M.\t\r \u00a0G.\t\r \u00a0Winter,\t\r \u00a0B.\t\r \u00a0P.\t\r \u00a0Butler,\t\r \u00a0D.\t\r \u00a0L.\t\r \u00a0Huseby,\t\r \u00a0R.\t\r \u00a0W.\t\r \u00a0Crawford,\t\r \u00a0J.\t\r \u00a0M.\t\r \u00a0Russell,\t\r \u00a0C.\t\r \u00a0L.\t\r \u00a0Bevins,\t\r \u00a0L.\t\r \u00a0G.\t\r \u00a0Adams,\t\r \u00a0R.\t\r \u00a0M.\t\r \u00a0Tsolis,\t\r \u00a0J.\t\r \u00a0R.\t\r \u00a0Roth,\t\r \u00a0A.\t\r \u00a0J.\t\r \u00a0Baumler,\t\r \u00a0Gut\t\r \u00a0inflammation\t\r \u00a0provides\t\r \u00a0a\t\r \u00a0respiratory\t\r \u00a0electron\t\r \u00a0acceptor\t\r \u00a0for\t\r \u00a0Salmonella.\t\r \u00a0Nature\t\r \u00a0467,\t\r \u00a0426-\u00ad\u2010429\t\r \u00a0(2010).\t\r \u00a0192.\t\r \u00a0 A.\t\r \u00a0K.\t\r \u00a0Criss,\t\r \u00a0M.\t\r \u00a0Silva,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Casanova,\t\r \u00a0B.\t\r \u00a0A.\t\r \u00a0McCormick,\t\r \u00a0Regulation\t\r \u00a0of\t\r \u00a0Salmonella-\u00ad\u2010induced\t\r \u00a0neutrophil\t\r \u00a0transmigration\t\r \u00a0by\t\r \u00a0epithelial\t\r \u00a0ADP-\u00ad\u2010ribosylation\t\r \u00a0factor\t\r \u00a06.\t\r \u00a0J\t\r \u00a0 Biol\t\r \u00a0Chem\t\r \u00a0276,\t\r \u00a048431-\u00ad\u201048439\t\r \u00a0(2001).\t\r \u00a0193.\t\r \u00a0 M.\t\r \u00a0Silva,\t\r \u00a0C.\t\r \u00a0Song,\t\r \u00a0W.\t\r \u00a0J.\t\r \u00a0Nadeau,\t\r \u00a0J.\t\r \u00a0B.\t\r \u00a0Matthews,\t\r \u00a0B.\t\r \u00a0A.\t\r \u00a0McCormick,\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0SipA-\u00ad\u2010induced\t\r \u00a0neutrophil\t\r \u00a0transepithelial\t\r \u00a0migration:\t\r \u00a0involvement\t\r \u00a0of\t\r \u00a0a\t\r \u00a0PKC-\u00ad\u2010alpha-\u00ad\u2010dependent\t\r \u00a0signal\t\r \u00a0transduction\t\r \u00a0pathway.\t\r \u00a0Am\t\r \u00a0J\t\r \u00a0 Physiol\t\r \u00a0Gastrointest\t\r \u00a0Liver\t\r \u00a0Physiol\t\r \u00a0286,\t\r \u00a0G1024-\u00ad\u20101031\t\r \u00a0(2004).\t\r \u00a0194.\t\r \u00a0 J.\t\r \u00a0F.\t\r \u00a0Figueiredo,\t\r \u00a0S.\t\r \u00a0D.\t\r \u00a0Lawhon,\t\r \u00a0K.\t\r \u00a0Gokulan,\t\r \u00a0S.\t\r \u00a0Khare,\t\r \u00a0M.\t\r \u00a0Raffatellu,\t\r \u00a0R.\t\r \u00a0M.\t\r \u00a0Tsolis,\t\r \u00a0A.\t\r \u00a0J.\t\r \u00a0Baumler,\t\r \u00a0B.\t\r \u00a0A.\t\r \u00a0McCormick,\t\r \u00a0L.\t\r \u00a0G.\t\r \u00a0Adams,\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0Typhimurium\t\r \u00a0SipA\t\r \u00a0induces\t\r \u00a0CXC-\u00ad\u2010chemokine\t\r \u00a0expression\t\r \u00a0through\t\r \u00a0p38MAPK\t\r \u00a0and\t\r \u00a0JUN\t\r \u00a0pathways.\t\r \u00a0Microbes\t\r \u00a0Infect\t\r \u00a011,\t\r \u00a0302-\u00ad\u2010310\t\r \u00a0(2009).\t\r \u00a0195.\t\r \u00a0 N.\t\r \u00a0Le\t\r \u00a0Novere,\t\r \u00a0M.\t\r \u00a0Hucka,\t\r \u00a0H.\t\r \u00a0Mi,\t\r \u00a0S.\t\r \u00a0Moodie,\t\r \u00a0F.\t\r \u00a0Schreiber,\t\r \u00a0A.\t\r \u00a0Sorokin,\t\r \u00a0E.\t\r \u00a0Demir,\t\r \u00a0K.\t\r \u00a0Wegner,\t\r \u00a0M.\t\r \u00a0I.\t\r \u00a0Aladjem,\t\r \u00a0S.\t\r \u00a0M.\t\r \u00a0Wimalaratne,\t\r \u00a0F.\t\r \u00a0T.\t\r \u00a0Bergman,\t\r \u00a0R.\t\r \u00a0Gauges,\t\r \u00a0P.\t\r \u00a0Ghazal,\t\r \u00a0H.\t\r \u00a0Kawaji,\t\r \u00a0L.\t\r \u00a0Li,\t\r \u00a0Y.\t\r \u00a0Matsuoka,\t\r \u00a0A.\t\r \u00a0Villeger,\t\r \u00a0S.\t\r \u00a0E.\t\r \u00a0Boyd,\t\r \u00a0L.\t\r \u00a0Calzone,\t\r \u00a0M.\t\r \u00a0Courtot,\t\r \u00a0U.\t\r \u00a0Dogrusoz,\t\r \u00a0T.\t\r \u00a0C.\t\r \u00a0Freeman,\t\r \u00a0A.\t\r \u00a0Funahashi,\t\r \u00a0S.\t\r \u00a0Ghosh,\t\r \u00a0A.\t\r \u00a0Jouraku,\t\r \u00a0S.\t\r \u00a0Kim,\t\r \u00a0F.\t\r \u00a0Kolpakov,\t\r \u00a0A.\t\r \u00a0Luna,\t\r \u00a0S.\t\r \u00a0Sahle,\t\r \u00a0E.\t\r \u00a0Schmidt,\t\r \u00a0S.\t\r \u00a0Watterson,\t\r \u00a0G.\t\r \u00a0Wu,\t\r \u00a0I.\t\r \u00a0Goryanin,\t\r \u00a0D.\t\r \u00a0B.\t\r \u00a0Kell,\t\r \u00a0C.\t\r \u00a0Sander,\t\r \u00a0H.\t\r \u00a0Sauro,\t\r \u00a0J.\t\r \u00a0L.\t\r \u00a0Snoep,\t\r \u00a0K.\t\r \u00a0Kohn,\t\r \u00a0H.\t\r \u00a0Kitano,\t\r \u00a0The\t\r \u00a0Systems\t\r \u00a0Biology\t\r \u00a0Graphical\t\r \u00a0Notation.\t\r \u00a0Nat\t\r \u00a0Biotechnol\t\r \u00a027,\t\r \u00a0735-\u00ad\u2010741\t\r \u00a0(2009).\t\r \u00a0196.\t\r \u00a0 T.\t\r \u00a0Kubori,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Temporal\t\r \u00a0regulation\t\r \u00a0of\t\r \u00a0salmonella\t\r \u00a0virulence\t\r \u00a0effector\t\r \u00a0function\t\r \u00a0by\t\r \u00a0proteasome-\u00ad\u2010dependent\t\r \u00a0protein\t\r \u00a0degradation.\t\r \u00a0Cell\t\r \u00a0115,\t\r \u00a0333-\u00ad\u2010342\t\r \u00a0(2003).\t\r \u00a0197.\t\r \u00a0 J.\t\r \u00a0C.\t\r \u00a0Patel,\t\r \u00a0K.\t\r \u00a0Hueffer,\t\r \u00a0T.\t\r \u00a0T.\t\r \u00a0Lam,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Diversification\t\r \u00a0of\t\r \u00a0a\t\r \u00a0Salmonella\t\r \u00a0virulence\t\r \u00a0protein\t\r \u00a0function\t\r \u00a0by\t\r \u00a0ubiquitin-\u00ad\u2010dependent\t\r \u00a0differential\t\r \u00a0localization.\t\r \u00a0 Cell\t\r \u00a0137,\t\r \u00a0283-\u00ad\u2010294\t\r \u00a0(2009).\t\r \u00a0198.\t\r \u00a0 L.\t\r \u00a0A.\t\r \u00a0Knodler,\t\r \u00a0S.\t\r \u00a0Winfree,\t\r \u00a0D.\t\r \u00a0Drecktrah,\t\r \u00a0R.\t\r \u00a0Ireland,\t\r \u00a0O.\t\r \u00a0Steele-\u00ad\u2010Mortimer,\t\r \u00a0Ubiquitination\t\r \u00a0of\t\r \u00a0the\t\r \u00a0bacterial\t\r \u00a0inositol\t\r \u00a0phosphatase,\t\r \u00a0SopB,\t\r \u00a0regulates\t\r \u00a0its\t\r \u00a0biological\t\r \u00a0activity\t\r \u00a0at\t\r \u00a0the\t\r \u00a0plasma\t\r \u00a0membrane.\t\r \u00a0Cell\t\r \u00a0Microbiol\t\r \u00a011,\t\r \u00a01652-\u00ad\u20101670\t\r \u00a0(2009).\t\r \u00a0199.\t\r \u00a0 M.\t\r \u00a0A.\t\r \u00a0Bakowski,\t\r \u00a0V.\t\r \u00a0Braun,\t\r \u00a0G.\t\r \u00a0Y.\t\r \u00a0Lam,\t\r \u00a0T.\t\r \u00a0Yeung,\t\r \u00a0W.\t\r \u00a0Do\t\r \u00a0Heo,\t\r \u00a0T.\t\r \u00a0Meyer,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0S.\t\r \u00a0Grinstein,\t\r \u00a0J.\t\r \u00a0H.\t\r \u00a0Brumell,\t\r \u00a0The\t\r \u00a0phosphoinositide\t\r \u00a0phosphatase\t\r \u00a0SopB\t\r \u00a0manipulates\t\r \u00a0membrane\t\r \u00a0surface\t\r \u00a0charge\t\r \u00a0and\t\r \u00a0trafficking\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Salmonella-\u00ad\u2010containing\t\r \u00a0vacuole.\t\r \u00a0Cell\t\r \u00a0Host\t\r \u00a0Microbe\t\r \u00a07,\t\r \u00a0453-\u00ad\u2010462\t\r \u00a0(2010).\t\r \u00a0200.\t\r \u00a0 J.\t\r \u00a0D.\t\r \u00a0Dukes,\t\r \u00a0H.\t\r \u00a0Lee,\t\r \u00a0R.\t\r \u00a0Hagen,\t\r \u00a0B.\t\r \u00a0J.\t\r \u00a0Reaves,\t\r \u00a0A.\t\r \u00a0N.\t\r \u00a0Layton,\t\r \u00a0E.\t\r \u00a0E.\t\r \u00a0Galyov,\t\r \u00a0P.\t\r \u00a0Whitley,\t\r \u00a0The\t\r \u00a0secreted\t\r \u00a0Salmonella\t\r \u00a0dublin\t\r \u00a0phosphoinositide\t\r \u00a0phosphatase,\t\r \u00a0SopB,\t\r \u00a0 \t\r \u00a0\t\r \u00a0 157\t\r \u00a0 localizes\t\r \u00a0to\t\r \u00a0PtdIns(3)P-\u00ad\u2010containing\t\r \u00a0endosomes\t\r \u00a0and\t\r \u00a0perturbs\t\r \u00a0normal\t\r \u00a0endosome\t\r \u00a0to\t\r \u00a0lysosome\t\r \u00a0trafficking.\t\r \u00a0Biochem\t\r \u00a0J\t\r \u00a0395,\t\r \u00a0239-\u00ad\u2010247\t\r \u00a0(2006).\t\r \u00a0201.\t\r \u00a0 D.\t\r \u00a0Drecktrah,\t\r \u00a0L.\t\r \u00a0A.\t\r \u00a0Knodler,\t\r \u00a0K.\t\r \u00a0Galbraith,\t\r \u00a0O.\t\r \u00a0Steele-\u00ad\u2010Mortimer,\t\r \u00a0The\t\r \u00a0Salmonella\t\r \u00a0SPI1\t\r \u00a0effector\t\r \u00a0SopB\t\r \u00a0stimulates\t\r \u00a0nitric\t\r \u00a0oxide\t\r \u00a0production\t\r \u00a0long\t\r \u00a0after\t\r \u00a0invasion.\t\r \u00a0Cell\t\r \u00a0 Microbiol\t\r \u00a07,\t\r \u00a0105-\u00ad\u2010113\t\r \u00a0(2005).\t\r \u00a0202.\t\r \u00a0 R.\t\r \u00a0J.\t\r \u00a0Cain,\t\r \u00a0R.\t\r \u00a0D.\t\r \u00a0Hayward,\t\r \u00a0V.\t\r \u00a0Koronakis,\t\r \u00a0Deciphering\t\r \u00a0interplay\t\r \u00a0between\t\r \u00a0Salmonella\t\r \u00a0invasion\t\r \u00a0effectors.\t\r \u00a0PLoS\t\r \u00a0Pathog\t\r \u00a04,\t\r \u00a0e1000037\t\r \u00a0(2008).\t\r \u00a0203.\t\r \u00a0 J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0H.\t\r \u00a0Wolf-\u00ad\u2010Watz,\t\r \u00a0Protein\t\r \u00a0delivery\t\r \u00a0into\t\r \u00a0eukaryotic\t\r \u00a0cells\t\r \u00a0by\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0machines.\t\r \u00a0Nature\t\r \u00a0444,\t\r \u00a0567-\u00ad\u2010573\t\r \u00a0(2006).\t\r \u00a0204.\t\r \u00a0 M.\t\r \u00a0F.\t\r \u00a0Feldman,\t\r \u00a0G.\t\r \u00a0R.\t\r \u00a0Cornelis,\t\r \u00a0The\t\r \u00a0multitalented\t\r \u00a0type\t\r \u00a0III\t\r \u00a0chaperones:\t\r \u00a0all\t\r \u00a0you\t\r \u00a0can\t\r \u00a0do\t\r \u00a0with\t\r \u00a015\t\r \u00a0kDa.\t\r \u00a0FEMS\t\r \u00a0Microbiol\t\r \u00a0Lett\t\r \u00a0219,\t\r \u00a0151-\u00ad\u2010158\t\r \u00a0(2003).\t\r \u00a0205.\t\r \u00a0 C.\t\r \u00a0E.\t\r \u00a0Stebbins,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Priming\t\r \u00a0virulence\t\r \u00a0factors\t\r \u00a0for\t\r \u00a0delivery\t\r \u00a0into\t\r \u00a0the\t\r \u00a0host.\t\r \u00a0 Nat\t\r \u00a0Rev\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Biol\t\r \u00a04,\t\r \u00a0738-\u00ad\u2010743\t\r \u00a0(2003).\t\r \u00a0206.\t\r \u00a0 M.\t\r \u00a0Lilic,\t\r \u00a0M.\t\r \u00a0Vujanac,\t\r \u00a0C.\t\r \u00a0E.\t\r \u00a0Stebbins,\t\r \u00a0A\t\r \u00a0common\t\r \u00a0structural\t\r \u00a0motif\t\r \u00a0in\t\r \u00a0the\t\r \u00a0binding\t\r \u00a0of\t\r \u00a0virulence\t\r \u00a0factors\t\r \u00a0to\t\r \u00a0bacterial\t\r \u00a0secretion\t\r \u00a0chaperones.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a021,\t\r \u00a0653-\u00ad\u2010664\t\r \u00a0(2006).\t\r \u00a0207.\t\r \u00a0 Y.\t\r \u00a0Luo,\t\r \u00a0M.\t\r \u00a0G.\t\r \u00a0Bertero,\t\r \u00a0E.\t\r \u00a0A.\t\r \u00a0Frey,\t\r \u00a0R.\t\r \u00a0A.\t\r \u00a0Pfuetzner,\t\r \u00a0M.\t\r \u00a0R.\t\r \u00a0Wenk,\t\r \u00a0L.\t\r \u00a0Creagh,\t\r \u00a0S.\t\r \u00a0L.\t\r \u00a0Marcus,\t\r \u00a0D.\t\r \u00a0Lim,\t\r \u00a0F.\t\r \u00a0Sicheri,\t\r \u00a0C.\t\r \u00a0Kay,\t\r \u00a0C.\t\r \u00a0Haynes,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0N.\t\r \u00a0C.\t\r \u00a0Strynadka,\t\r \u00a0Structural\t\r \u00a0and\t\r \u00a0biochemical\t\r \u00a0characterization\t\r \u00a0of\t\r \u00a0the\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0chaperones\t\r \u00a0CesT\t\r \u00a0and\t\r \u00a0SigE.\t\r \u00a0Nat\t\r \u00a0Struct\t\r \u00a0Biol\t\r \u00a08,\t\r \u00a01031-\u00ad\u20101036\t\r \u00a0(2001).\t\r \u00a0208.\t\r \u00a0 C.\t\r \u00a0E.\t\r \u00a0Stebbins,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Maintenance\t\r \u00a0of\t\r \u00a0an\t\r \u00a0unfolded\t\r \u00a0polypeptide\t\r \u00a0by\t\r \u00a0a\t\r \u00a0cognate\t\r \u00a0chaperone\t\r \u00a0in\t\r \u00a0bacterial\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion.\t\r \u00a0Nature\t\r \u00a0414,\t\r \u00a077-\u00ad\u201081\t\r \u00a0(2001).\t\r \u00a0209.\t\r \u00a0 S.\t\r \u00a0C.\t\r \u00a0Birtalan,\t\r \u00a0R.\t\r \u00a0M.\t\r \u00a0Phillips,\t\r \u00a0P.\t\r \u00a0Ghosh,\t\r \u00a0Three-\u00ad\u2010dimensional\t\r \u00a0secretion\t\r \u00a0signals\t\r \u00a0in\t\r \u00a0chaperone-\u00ad\u2010effector\t\r \u00a0complexes\t\r \u00a0of\t\r \u00a0bacterial\t\r \u00a0pathogens.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a09,\t\r \u00a0971-\u00ad\u2010980\t\r \u00a0(2002).\t\r \u00a0210.\t\r \u00a0 S.\t\r \u00a0C.\t\r \u00a0Tucker,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Complex\t\r \u00a0function\t\r \u00a0for\t\r \u00a0SicA,\t\r \u00a0a\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0typhimurium\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion-\u00ad\u2010associated\t\r \u00a0chaperone.\t\r \u00a0J\t\r \u00a0Bacteriol\t\r \u00a0 182,\t\r \u00a02262-\u00ad\u20102268\t\r \u00a0(2000).\t\r \u00a0211.\t\r \u00a0 Y.\t\r \u00a0Fu,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Identification\t\r \u00a0of\t\r \u00a0a\t\r \u00a0specific\t\r \u00a0chaperone\t\r \u00a0for\t\r \u00a0SptP,\t\r \u00a0a\t\r \u00a0substrate\t\r \u00a0of\t\r \u00a0the\t\r \u00a0centisome\t\r \u00a063\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0system\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0typhimurium.\t\r \u00a0J\t\r \u00a0 Bacteriol\t\r \u00a0180,\t\r \u00a03393-\u00ad\u20103399\t\r \u00a0(1998).\t\r \u00a0212.\t\r \u00a0 K.\t\r \u00a0H.\t\r \u00a0Darwin,\t\r \u00a0L.\t\r \u00a0S.\t\r \u00a0Robinson,\t\r \u00a0V.\t\r \u00a0L.\t\r \u00a0Miller,\t\r \u00a0SigE\t\r \u00a0is\t\r \u00a0a\t\r \u00a0chaperone\t\r \u00a0for\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0Typhimurium\t\r \u00a0invasion\t\r \u00a0protein\t\r \u00a0SigD.\t\r \u00a0J\t\r \u00a0Bacteriol\t\r \u00a0 183,\t\r \u00a01452-\u00ad\u20101454\t\r \u00a0(2001).\t\r \u00a0213.\t\r \u00a0 L.\t\r \u00a0A.\t\r \u00a0Knodler,\t\r \u00a0M.\t\r \u00a0Bertero,\t\r \u00a0C.\t\r \u00a0Yip,\t\r \u00a0N.\t\r \u00a0C.\t\r \u00a0Strynadka,\t\r \u00a0O.\t\r \u00a0Steele-\u00ad\u2010Mortimer,\t\r \u00a0Structure-\u00ad\u2010based\t\r \u00a0mutagenesis\t\r \u00a0of\t\r \u00a0SigE\t\r \u00a0verifies\t\r \u00a0the\t\r \u00a0importance\t\r \u00a0of\t\r \u00a0hydrophobic\t\r \u00a0and\t\r \u00a0electrostatic\t\r \u00a0residues\t\r \u00a0in\t\r \u00a0type\t\r \u00a0III\t\r \u00a0chaperone\t\r \u00a0function.\t\r \u00a0Mol\t\r \u00a0Microbiol\t\r \u00a062,\t\r \u00a0928-\u00ad\u2010940\t\r \u00a0(2006).\t\r \u00a0214.\t\r \u00a0 P.\t\r \u00a0A.\t\r \u00a0Bronstein,\t\r \u00a0E.\t\r \u00a0A.\t\r \u00a0Miao,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0InvB\t\r \u00a0is\t\r \u00a0a\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0chaperone\t\r \u00a0specific\t\r \u00a0for\t\r \u00a0SspA.\t\r \u00a0J\t\r \u00a0Bacteriol\t\r \u00a0182,\t\r \u00a06638-\u00ad\u20106644\t\r \u00a0(2000).\t\r \u00a0215.\t\r \u00a0 K.\t\r \u00a0Ehrbar,\t\r \u00a0A.\t\r \u00a0Friebel,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0W.\t\r \u00a0D.\t\r \u00a0Hardt,\t\r \u00a0Role\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0pathogenicity\t\r \u00a0island\t\r \u00a01\t\r \u00a0(SPI-\u00ad\u20101)\t\r \u00a0protein\t\r \u00a0InvB\t\r \u00a0in\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0of\t\r \u00a0SopE\t\r \u00a0and\t\r \u00a0SopE2,\t\r \u00a0two\t\r \u00a0Salmonella\t\r \u00a0effector\t\r \u00a0proteins\t\r \u00a0encoded\t\r \u00a0outside\t\r \u00a0of\t\r \u00a0SPI-\u00ad\u20101.\t\r \u00a0J\t\r \u00a0Bacteriol\t\r \u00a0 185,\t\r \u00a06950-\u00ad\u20106967\t\r \u00a0(2003).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 158\t\r \u00a0 216.\t\r \u00a0 S.\t\r \u00a0H.\t\r \u00a0Lee,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0InvB\t\r \u00a0is\t\r \u00a0a\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion-\u00ad\u2010associated\t\r \u00a0chaperone\t\r \u00a0for\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0effector\t\r \u00a0protein\t\r \u00a0SopE.\t\r \u00a0J\t\r \u00a0Bacteriol\t\r \u00a0185,\t\r \u00a07279-\u00ad\u20107284\t\r \u00a0(2003).\t\r \u00a0217.\t\r \u00a0 K.\t\r \u00a0Ehrbar,\t\r \u00a0S.\t\r \u00a0Hapfelmeier,\t\r \u00a0B.\t\r \u00a0Stecher,\t\r \u00a0W.\t\r \u00a0D.\t\r \u00a0Hardt,\t\r \u00a0InvB\t\r \u00a0is\t\r \u00a0required\t\r \u00a0for\t\r \u00a0type\t\r \u00a0III-\u00ad\u2010dependent\t\r \u00a0secretion\t\r \u00a0of\t\r \u00a0SopA\t\r \u00a0in\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0Typhimurium.\t\r \u00a0J\t\r \u00a0Bacteriol\t\r \u00a0186,\t\r \u00a01215-\u00ad\u20101219\t\r \u00a0(2004).\t\r \u00a0218.\t\r \u00a0 Y.\t\r \u00a0Akeda,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Chaperone\t\r \u00a0release\t\r \u00a0and\t\r \u00a0unfolding\t\r \u00a0of\t\r \u00a0substrates\t\r \u00a0in\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion.\t\r \u00a0Nature\t\r \u00a0437,\t\r \u00a0911-\u00ad\u2010915\t\r \u00a0(2005).\t\r \u00a0219.\t\r \u00a0 M.\t\r \u00a0Hensel,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Shea,\t\r \u00a0B.\t\r \u00a0Raupach,\t\r \u00a0D.\t\r \u00a0Monack,\t\r \u00a0S.\t\r \u00a0Falkow,\t\r \u00a0C.\t\r \u00a0Gleeson,\t\r \u00a0T.\t\r \u00a0Kubo,\t\r \u00a0D.\t\r \u00a0W.\t\r \u00a0Holden,\t\r \u00a0Functional\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0ssaJ\t\r \u00a0and\t\r \u00a0the\t\r \u00a0ssaK\/U\t\r \u00a0operon,\t\r \u00a013\t\r \u00a0genes\t\r \u00a0encoding\t\r \u00a0components\t\r \u00a0of\t\r \u00a0the\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0apparatus\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0Pathogenicity\t\r \u00a0Island\t\r \u00a02.\t\r \u00a0Mol\t\r \u00a0Microbiol\t\r \u00a024,\t\r \u00a0155-\u00ad\u2010167\t\r \u00a0(1997).\t\r \u00a0220.\t\r \u00a0 J.\t\r \u00a0Deiwick,\t\r \u00a0T.\t\r \u00a0Nikolaus,\t\r \u00a0S.\t\r \u00a0Erdogan,\t\r \u00a0M.\t\r \u00a0Hensel,\t\r \u00a0Environmental\t\r \u00a0regulation\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0pathogenicity\t\r \u00a0island\t\r \u00a02\t\r \u00a0gene\t\r \u00a0expression.\t\r \u00a0Mol\t\r \u00a0Microbiol\t\r \u00a031,\t\r \u00a01759-\u00ad\u20101773\t\r \u00a0(1999).\t\r \u00a0221.\t\r \u00a0 A.\t\r \u00a0K.\t\r \u00a0Lee,\t\r \u00a0C.\t\r \u00a0S.\t\r \u00a0Detweiler,\t\r \u00a0S.\t\r \u00a0Falkow,\t\r \u00a0OmpR\t\r \u00a0regulates\t\r \u00a0the\t\r \u00a0two-\u00ad\u2010component\t\r \u00a0system\t\r \u00a0SsrA-\u00ad\u2010ssrB\t\r \u00a0in\t\r \u00a0Salmonella\t\r \u00a0pathogenicity\t\r \u00a0island\t\r \u00a02.\t\r \u00a0J\t\r \u00a0Bacteriol\t\r \u00a0182,\t\r \u00a0771-\u00ad\u2010781\t\r \u00a0(2000).\t\r \u00a0222.\t\r \u00a0 J.\t\r \u00a0Garmendia,\t\r \u00a0C.\t\r \u00a0R.\t\r \u00a0Beuzon,\t\r \u00a0J.\t\r \u00a0Ruiz-\u00ad\u2010Albert,\t\r \u00a0D.\t\r \u00a0W.\t\r \u00a0Holden,\t\r \u00a0The\t\r \u00a0roles\t\r \u00a0of\t\r \u00a0SsrA-\u00ad\u2010SsrB\t\r \u00a0and\t\r \u00a0OmpR-\u00ad\u2010EnvZ\t\r \u00a0in\t\r \u00a0the\t\r \u00a0regulation\t\r \u00a0of\t\r \u00a0genes\t\r \u00a0encoding\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0SPI-\u00ad\u20102\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0system.\t\r \u00a0Microbiology\t\r \u00a0149,\t\r \u00a02385-\u00ad\u20102396\t\r \u00a0(2003).\t\r \u00a0223.\t\r \u00a0 X.\t\r \u00a0Feng,\t\r \u00a0R.\t\r \u00a0Oropeza,\t\r \u00a0L.\t\r \u00a0J.\t\r \u00a0Kenney,\t\r \u00a0Dual\t\r \u00a0regulation\t\r \u00a0by\t\r \u00a0phospho-\u00ad\u2010OmpR\t\r \u00a0of\t\r \u00a0ssrA\/B\t\r \u00a0gene\t\r \u00a0expression\t\r \u00a0in\t\r \u00a0Salmonella\t\r \u00a0pathogenicity\t\r \u00a0island\t\r \u00a02.\t\r \u00a0Mol\t\r \u00a0Microbiol\t\r \u00a048,\t\r \u00a01131-\u00ad\u20101143\t\r \u00a0(2003).\t\r \u00a0224.\t\r \u00a0 J.\t\r \u00a0J.\t\r \u00a0Bijlsma,\t\r \u00a0E.\t\r \u00a0A.\t\r \u00a0Groisman,\t\r \u00a0The\t\r \u00a0PhoP\/PhoQ\t\r \u00a0system\t\r \u00a0controls\t\r \u00a0the\t\r \u00a0intramacrophage\t\r \u00a0type\t\r \u00a0three\t\r \u00a0secretion\t\r \u00a0system\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0enterica.\t\r \u00a0Mol\t\r \u00a0 Microbiol\t\r \u00a057,\t\r \u00a085-\u00ad\u201096\t\r \u00a0(2005).\t\r \u00a0225.\t\r \u00a0 W.\t\r \u00a0W.\t\r \u00a0Navarre,\t\r \u00a0T.\t\r \u00a0A.\t\r \u00a0Halsey,\t\r \u00a0D.\t\r \u00a0Walthers,\t\r \u00a0J.\t\r \u00a0Frye,\t\r \u00a0M.\t\r \u00a0McClelland,\t\r \u00a0J.\t\r \u00a0L.\t\r \u00a0Potter,\t\r \u00a0L.\t\r \u00a0J.\t\r \u00a0Kenney,\t\r \u00a0J.\t\r \u00a0S.\t\r \u00a0Gunn,\t\r \u00a0F.\t\r \u00a0C.\t\r \u00a0Fang,\t\r \u00a0S.\t\r \u00a0J.\t\r \u00a0Libby,\t\r \u00a0Co-\u00ad\u2010regulation\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0genes\t\r \u00a0required\t\r \u00a0for\t\r \u00a0virulence\t\r \u00a0and\t\r \u00a0resistance\t\r \u00a0to\t\r \u00a0antimicrobial\t\r \u00a0peptides\t\r \u00a0by\t\r \u00a0SlyA\t\r \u00a0and\t\r \u00a0PhoP\/PhoQ.\t\r \u00a0Mol\t\r \u00a0Microbiol\t\r \u00a056,\t\r \u00a0492-\u00ad\u2010508\t\r \u00a0(2005).\t\r \u00a0226.\t\r \u00a0 C.\t\r \u00a0R.\t\r \u00a0Beuzon,\t\r \u00a0K.\t\r \u00a0E.\t\r \u00a0Unsworth,\t\r \u00a0D.\t\r \u00a0W.\t\r \u00a0Holden,\t\r \u00a0In\t\r \u00a0vivo\t\r \u00a0genetic\t\r \u00a0analysis\t\r \u00a0indicates\t\r \u00a0that\t\r \u00a0PhoP-\u00ad\u2010PhoQ\t\r \u00a0and\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0pathogenicity\t\r \u00a0island\t\r \u00a02\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0system\t\r \u00a0contribute\t\r \u00a0independently\t\r \u00a0to\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0Typhimurium\t\r \u00a0virulence.\t\r \u00a0Infect\t\r \u00a0Immun\t\r \u00a069,\t\r \u00a07254-\u00ad\u20107261\t\r \u00a0(2001).\t\r \u00a0227.\t\r \u00a0 D.\t\r \u00a0Walthers,\t\r \u00a0R.\t\r \u00a0K.\t\r \u00a0Carroll,\t\r \u00a0W.\t\r \u00a0W.\t\r \u00a0Navarre,\t\r \u00a0S.\t\r \u00a0J.\t\r \u00a0Libby,\t\r \u00a0F.\t\r \u00a0C.\t\r \u00a0Fang,\t\r \u00a0L.\t\r \u00a0J.\t\r \u00a0Kenney,\t\r \u00a0The\t\r \u00a0response\t\r \u00a0regulator\t\r \u00a0SsrB\t\r \u00a0activates\t\r \u00a0expression\t\r \u00a0of\t\r \u00a0diverse\t\r \u00a0Salmonella\t\r \u00a0pathogenicity\t\r \u00a0island\t\r \u00a02\t\r \u00a0promoters\t\r \u00a0and\t\r \u00a0counters\t\r \u00a0silencing\t\r \u00a0by\t\r \u00a0the\t\r \u00a0nucleoid-\u00ad\u2010associated\t\r \u00a0protein\t\r \u00a0H-\u00ad\u2010NS.\t\r \u00a0Mol\t\r \u00a0Microbiol\t\r \u00a065,\t\r \u00a0477-\u00ad\u2010493\t\r \u00a0(2007).\t\r \u00a0228.\t\r \u00a0 M.\t\r \u00a0J.\t\r \u00a0Worley,\t\r \u00a0K.\t\r \u00a0H.\t\r \u00a0Ching,\t\r \u00a0F.\t\r \u00a0Heffron,\t\r \u00a0Salmonella\t\r \u00a0SsrB\t\r \u00a0activates\t\r \u00a0a\t\r \u00a0global\t\r \u00a0regulon\t\r \u00a0of\t\r \u00a0horizontally\t\r \u00a0acquired\t\r \u00a0genes.\t\r \u00a0Mol\t\r \u00a0Microbiol\t\r \u00a036,\t\r \u00a0749-\u00ad\u2010761\t\r \u00a0(2000).\t\r \u00a0229.\t\r \u00a0 D.\t\r \u00a0Chakravortty,\t\r \u00a0M.\t\r \u00a0Rohde,\t\r \u00a0L.\t\r \u00a0Jager,\t\r \u00a0J.\t\r \u00a0Deiwick,\t\r \u00a0M.\t\r \u00a0Hensel,\t\r \u00a0Formation\t\r \u00a0of\t\r \u00a0a\t\r \u00a0novel\t\r \u00a0surface\t\r \u00a0structure\t\r \u00a0encoded\t\r \u00a0by\t\r \u00a0Salmonella\t\r \u00a0Pathogenicity\t\r \u00a0Island\t\r \u00a02.\t\r \u00a0EMBO\t\r \u00a0 J\t\r \u00a024,\t\r \u00a02043-\u00ad\u20102052\t\r \u00a0(2005).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 159\t\r \u00a0 230.\t\r \u00a0 T.\t\r \u00a0Nikolaus,\t\r \u00a0J.\t\r \u00a0Deiwick,\t\r \u00a0C.\t\r \u00a0Rappl,\t\r \u00a0J.\t\r \u00a0A.\t\r \u00a0Freeman,\t\r \u00a0W.\t\r \u00a0Schroder,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0M.\t\r \u00a0Hensel,\t\r \u00a0SseBCD\t\r \u00a0proteins\t\r \u00a0are\t\r \u00a0secreted\t\r \u00a0by\t\r \u00a0the\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0system\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0pathogenicity\t\r \u00a0island\t\r \u00a02\t\r \u00a0and\t\r \u00a0function\t\r \u00a0as\t\r \u00a0a\t\r \u00a0translocon.\t\r \u00a0J\t\r \u00a0Bacteriol\t\r \u00a0 183,\t\r \u00a06036-\u00ad\u20106045\t\r \u00a0(2001).\t\r \u00a0231.\t\r \u00a0 X.\t\r \u00a0J.\t\r \u00a0Yu,\t\r \u00a0K.\t\r \u00a0McGourty,\t\r \u00a0M.\t\r \u00a0Liu,\t\r \u00a0K.\t\r \u00a0E.\t\r \u00a0Unsworth,\t\r \u00a0D.\t\r \u00a0W.\t\r \u00a0Holden,\t\r \u00a0pH\t\r \u00a0sensing\t\r \u00a0by\t\r \u00a0intracellular\t\r \u00a0Salmonella\t\r \u00a0induces\t\r \u00a0effector\t\r \u00a0translocation.\t\r \u00a0Science\t\r \u00a0328,\t\r \u00a01040-\u00ad\u20101043\t\r \u00a0(2010).\t\r \u00a0232.\t\r \u00a0 C.\t\r \u00a0R.\t\r \u00a0Beuzon,\t\r \u00a0S.\t\r \u00a0Meresse,\t\r \u00a0K.\t\r \u00a0E.\t\r \u00a0Unsworth,\t\r \u00a0J.\t\r \u00a0Ruiz-\u00ad\u2010Albert,\t\r \u00a0S.\t\r \u00a0Garvis,\t\r \u00a0S.\t\r \u00a0R.\t\r \u00a0Waterman,\t\r \u00a0T.\t\r \u00a0A.\t\r \u00a0Ryder,\t\r \u00a0E.\t\r \u00a0Boucrot,\t\r \u00a0D.\t\r \u00a0W.\t\r \u00a0Holden,\t\r \u00a0Salmonella\t\r \u00a0maintains\t\r \u00a0the\t\r \u00a0integrity\t\r \u00a0of\t\r \u00a0its\t\r \u00a0intracellular\t\r \u00a0vacuole\t\r \u00a0through\t\r \u00a0the\t\r \u00a0action\t\r \u00a0of\t\r \u00a0SifA.\t\r \u00a0EMBO\t\r \u00a0J\t\r \u00a019,\t\r \u00a03235-\u00ad\u20103249\t\r \u00a0(2000).\t\r \u00a0233.\t\r \u00a0 J.\t\r \u00a0H.\t\r \u00a0Brumell,\t\r \u00a0D.\t\r \u00a0L.\t\r \u00a0Goosney,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0SifA,\t\r \u00a0a\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secreted\t\r \u00a0effector\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0typhimurium,\t\r \u00a0directs\t\r \u00a0Salmonella-\u00ad\u2010induced\t\r \u00a0filament\t\r \u00a0(Sif)\t\r \u00a0formation\t\r \u00a0along\t\r \u00a0microtubules.\t\r \u00a0Traffic\t\r \u00a03,\t\r \u00a0407-\u00ad\u2010415\t\r \u00a0(2002).\t\r \u00a0234.\t\r \u00a0 A.\t\r \u00a0T.\t\r \u00a0Reinicke,\t\r \u00a0J.\t\r \u00a0L.\t\r \u00a0Hutchinson,\t\r \u00a0A.\t\r \u00a0I.\t\r \u00a0Magee,\t\r \u00a0P.\t\r \u00a0Mastroeni,\t\r \u00a0J.\t\r \u00a0Trowsdale,\t\r \u00a0A.\t\r \u00a0P.\t\r \u00a0Kelly,\t\r \u00a0A\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0effector\t\r \u00a0protein\t\r \u00a0SifA\t\r \u00a0is\t\r \u00a0modified\t\r \u00a0by\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0prenylation\t\r \u00a0and\t\r \u00a0S-\u00ad\u2010acylation\t\r \u00a0machinery.\t\r \u00a0J\t\r \u00a0Biol\t\r \u00a0Chem\t\r \u00a0280,\t\r \u00a014620-\u00ad\u201014627\t\r \u00a0(2005).\t\r \u00a0235.\t\r \u00a0 A.\t\r \u00a0Dumont,\t\r \u00a0E.\t\r \u00a0Boucrot,\t\r \u00a0S.\t\r \u00a0Drevensek,\t\r \u00a0V.\t\r \u00a0Daire,\t\r \u00a0J.\t\r \u00a0P.\t\r \u00a0Gorvel,\t\r \u00a0C.\t\r \u00a0Pous,\t\r \u00a0D.\t\r \u00a0W.\t\r \u00a0Holden,\t\r \u00a0S.\t\r \u00a0Meresse,\t\r \u00a0SKIP,\t\r \u00a0the\t\r \u00a0host\t\r \u00a0target\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0virulence\t\r \u00a0factor\t\r \u00a0SifA,\t\r \u00a0promotes\t\r \u00a0kinesin-\u00ad\u20101-\u00ad\u2010dependent\t\r \u00a0vacuolar\t\r \u00a0membrane\t\r \u00a0exchanges.\t\r \u00a0Traffic\t\r \u00a0 11,\t\r \u00a0899-\u00ad\u2010911\t\r \u00a0(2010).\t\r \u00a0236.\t\r \u00a0 L.\t\r \u00a0K.\t\r \u00a0Jackson,\t\r \u00a0P.\t\r \u00a0Nawabi,\t\r \u00a0C.\t\r \u00a0Hentea,\t\r \u00a0E.\t\r \u00a0A.\t\r \u00a0Roark,\t\r \u00a0K.\t\r \u00a0Haldar,\t\r \u00a0The\t\r \u00a0Salmonella\t\r \u00a0virulence\t\r \u00a0protein\t\r \u00a0SifA\t\r \u00a0is\t\r \u00a0a\t\r \u00a0G\t\r \u00a0protein\t\r \u00a0antagonist.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a0105,\t\r \u00a014141-\u00ad\u201014146\t\r \u00a0(2008).\t\r \u00a0237.\t\r \u00a0 M.\t\r \u00a0B.\t\r \u00a0Ohlson,\t\r \u00a0Z.\t\r \u00a0Huang,\t\r \u00a0N.\t\r \u00a0M.\t\r \u00a0Alto,\t\r \u00a0M.\t\r \u00a0P.\t\r \u00a0Blanc,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Dixon,\t\r \u00a0J.\t\r \u00a0Chai,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0Structure\t\r \u00a0and\t\r \u00a0function\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0SifA\t\r \u00a0indicate\t\r \u00a0that\t\r \u00a0its\t\r \u00a0interactions\t\r \u00a0with\t\r \u00a0SKIP,\t\r \u00a0SseJ,\t\r \u00a0and\t\r \u00a0RhoA\t\r \u00a0family\t\r \u00a0GTPases\t\r \u00a0induce\t\r \u00a0endosomal\t\r \u00a0tubulation.\t\r \u00a0Cell\t\r \u00a0Host\t\r \u00a0 Microbe\t\r \u00a04,\t\r \u00a0434-\u00ad\u2010446\t\r \u00a0(2008).\t\r \u00a0238.\t\r \u00a0 M.\t\r \u00a0B.\t\r \u00a0Ohlson,\t\r \u00a0K.\t\r \u00a0Fluhr,\t\r \u00a0C.\t\r \u00a0L.\t\r \u00a0Birmingham,\t\r \u00a0J.\t\r \u00a0H.\t\r \u00a0Brumell,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0SseJ\t\r \u00a0deacylase\t\r \u00a0activity\t\r \u00a0by\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0Typhimurium\t\r \u00a0promotes\t\r \u00a0virulence\t\r \u00a0in\t\r \u00a0mice.\t\r \u00a0Infect\t\r \u00a0Immun\t\r \u00a073,\t\r \u00a06249-\u00ad\u20106259\t\r \u00a0(2005).\t\r \u00a0239.\t\r \u00a0 J.\t\r \u00a0Ruiz-\u00ad\u2010Albert,\t\r \u00a0X.\t\r \u00a0J.\t\r \u00a0Yu,\t\r \u00a0C.\t\r \u00a0R.\t\r \u00a0Beuzon,\t\r \u00a0A.\t\r \u00a0N.\t\r \u00a0Blakey,\t\r \u00a0E.\t\r \u00a0E.\t\r \u00a0Galyov,\t\r \u00a0D.\t\r \u00a0W.\t\r \u00a0Holden,\t\r \u00a0Complementary\t\r \u00a0activities\t\r \u00a0of\t\r \u00a0SseJ\t\r \u00a0and\t\r \u00a0SifA\t\r \u00a0regulate\t\r \u00a0dynamics\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0vacuolar\t\r \u00a0membrane.\t\r \u00a0Mol\t\r \u00a0Microbiol\t\r \u00a044,\t\r \u00a0645-\u00ad\u2010661\t\r \u00a0(2002).\t\r \u00a0240.\t\r \u00a0 X.\t\r \u00a0Jiang,\t\r \u00a0O.\t\r \u00a0W.\t\r \u00a0Rossanese,\t\r \u00a0N.\t\r \u00a0F.\t\r \u00a0Brown,\t\r \u00a0S.\t\r \u00a0Kujat-\u00ad\u2010Choy,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0J.\t\r \u00a0H.\t\r \u00a0Brumell,\t\r \u00a0The\t\r \u00a0related\t\r \u00a0effector\t\r \u00a0proteins\t\r \u00a0SopD\t\r \u00a0and\t\r \u00a0SopD2\t\r \u00a0from\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0Typhimurium\t\r \u00a0contribute\t\r \u00a0to\t\r \u00a0virulence\t\r \u00a0during\t\r \u00a0systemic\t\r \u00a0infection\t\r \u00a0of\t\r \u00a0mice.\t\r \u00a0Mol\t\r \u00a0Microbiol\t\r \u00a054,\t\r \u00a01186-\u00ad\u20101198\t\r \u00a0(2004).\t\r \u00a0241.\t\r \u00a0 N.\t\r \u00a0Schroeder,\t\r \u00a0T.\t\r \u00a0Henry,\t\r \u00a0C.\t\r \u00a0de\t\r \u00a0Chastellier,\t\r \u00a0W.\t\r \u00a0Zhao,\t\r \u00a0A.\t\r \u00a0A.\t\r \u00a0Guilhon,\t\r \u00a0J.\t\r \u00a0P.\t\r \u00a0Gorvel,\t\r \u00a0S.\t\r \u00a0Meresse,\t\r \u00a0The\t\r \u00a0virulence\t\r \u00a0protein\t\r \u00a0SopD2\t\r \u00a0regulates\t\r \u00a0membrane\t\r \u00a0dynamics\t\r \u00a0of\t\r \u00a0Salmonella-\u00ad\u2010containing\t\r \u00a0vacuoles.\t\r \u00a0PLoS\t\r \u00a0Pathog\t\r \u00a06,\t\r \u00a0e1001002\t\r \u00a0(2010).\t\r \u00a0242.\t\r \u00a0 J.\t\r \u00a0Deiwick,\t\r \u00a0S.\t\r \u00a0P.\t\r \u00a0Salcedo,\t\r \u00a0E.\t\r \u00a0Boucrot,\t\r \u00a0S.\t\r \u00a0M.\t\r \u00a0Gilliland,\t\r \u00a0T.\t\r \u00a0Henry,\t\r \u00a0N.\t\r \u00a0Petermann,\t\r \u00a0S.\t\r \u00a0R.\t\r \u00a0Waterman,\t\r \u00a0J.\t\r \u00a0P.\t\r \u00a0Gorvel,\t\r \u00a0D.\t\r \u00a0W.\t\r \u00a0Holden,\t\r \u00a0S.\t\r \u00a0Meresse,\t\r \u00a0The\t\r \u00a0translocated\t\r \u00a0Salmonella\t\r \u00a0effector\t\r \u00a0proteins\t\r \u00a0SseF\t\r \u00a0and\t\r \u00a0SseG\t\r \u00a0interact\t\r \u00a0and\t\r \u00a0are\t\r \u00a0required\t\r \u00a0to\t\r \u00a0 \t\r \u00a0\t\r \u00a0 160\t\r \u00a0 establish\t\r \u00a0an\t\r \u00a0intracellular\t\r \u00a0replication\t\r \u00a0niche.\t\r \u00a0Infect\t\r \u00a0Immun\t\r \u00a074,\t\r \u00a06965-\u00ad\u20106972\t\r \u00a0(2006).\t\r \u00a0243.\t\r \u00a0 V.\t\r \u00a0Kuhle,\t\r \u00a0D.\t\r \u00a0Jackel,\t\r \u00a0M.\t\r \u00a0Hensel,\t\r \u00a0Effector\t\r \u00a0proteins\t\r \u00a0encoded\t\r \u00a0by\t\r \u00a0Salmonella\t\r \u00a0pathogenicity\t\r \u00a0island\t\r \u00a02\t\r \u00a0interfere\t\r \u00a0with\t\r \u00a0the\t\r \u00a0microtubule\t\r \u00a0cytoskeleton\t\r \u00a0after\t\r \u00a0translocation\t\r \u00a0into\t\r \u00a0host\t\r \u00a0cells.\t\r \u00a0Traffic\t\r \u00a05,\t\r \u00a0356-\u00ad\u2010370\t\r \u00a0(2004).\t\r \u00a0244.\t\r \u00a0 V.\t\r \u00a0Kuhle,\t\r \u00a0M.\t\r \u00a0Hensel,\t\r \u00a0SseF\t\r \u00a0and\t\r \u00a0SseG\t\r \u00a0are\t\r \u00a0translocated\t\r \u00a0effectors\t\r \u00a0of\t\r \u00a0the\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0system\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0pathogenicity\t\r \u00a0island\t\r \u00a02\t\r \u00a0that\t\r \u00a0modulate\t\r \u00a0aggregation\t\r \u00a0of\t\r \u00a0endosomal\t\r \u00a0compartments.\t\r \u00a0Cell\t\r \u00a0Microbiol\t\r \u00a04,\t\r \u00a0813-\u00ad\u2010824\t\r \u00a0(2002).\t\r \u00a0245.\t\r \u00a0 G.\t\r \u00a0L.\t\r \u00a0Abrahams,\t\r \u00a0P.\t\r \u00a0Muller,\t\r \u00a0M.\t\r \u00a0Hensel,\t\r \u00a0Functional\t\r \u00a0dissection\t\r \u00a0of\t\r \u00a0SseF,\t\r \u00a0a\t\r \u00a0type\t\r \u00a0III\t\r \u00a0effector\t\r \u00a0protein\t\r \u00a0involved\t\r \u00a0in\t\r \u00a0positioning\t\r \u00a0the\t\r \u00a0salmonella-\u00ad\u2010containing\t\r \u00a0vacuole.\t\r \u00a0 Traffic\t\r \u00a07,\t\r \u00a0950-\u00ad\u2010965\t\r \u00a0(2006).\t\r \u00a0246.\t\r \u00a0 S.\t\r \u00a0P.\t\r \u00a0Salcedo,\t\r \u00a0D.\t\r \u00a0W.\t\r \u00a0Holden,\t\r \u00a0SseG,\t\r \u00a0a\t\r \u00a0virulence\t\r \u00a0protein\t\r \u00a0that\t\r \u00a0targets\t\r \u00a0Salmonella\t\r \u00a0to\t\r \u00a0the\t\r \u00a0Golgi\t\r \u00a0network.\t\r \u00a0EMBO\t\r \u00a0J\t\r \u00a022,\t\r \u00a05003-\u00ad\u20105014\t\r \u00a0(2003).\t\r \u00a0247.\t\r \u00a0 L.\t\r \u00a0A.\t\r \u00a0Knodler,\t\r \u00a0O.\t\r \u00a0Steele-\u00ad\u2010Mortimer,\t\r \u00a0The\t\r \u00a0Salmonella\t\r \u00a0effector\t\r \u00a0PipB2\t\r \u00a0affects\t\r \u00a0late\t\r \u00a0endosome\/lysosome\t\r \u00a0distribution\t\r \u00a0to\t\r \u00a0mediate\t\r \u00a0Sif\t\r \u00a0extension.\t\r \u00a0Mol\t\r \u00a0Biol\t\r \u00a0Cell\t\r \u00a016,\t\r \u00a04108-\u00ad\u20104123\t\r \u00a0(2005).\t\r \u00a0248.\t\r \u00a0 T.\t\r \u00a0Henry,\t\r \u00a0C.\t\r \u00a0Couillault,\t\r \u00a0P.\t\r \u00a0Rockenfeller,\t\r \u00a0E.\t\r \u00a0Boucrot,\t\r \u00a0A.\t\r \u00a0Dumont,\t\r \u00a0N.\t\r \u00a0Schroeder,\t\r \u00a0A.\t\r \u00a0Hermant,\t\r \u00a0L.\t\r \u00a0A.\t\r \u00a0Knodler,\t\r \u00a0P.\t\r \u00a0Lecine,\t\r \u00a0O.\t\r \u00a0Steele-\u00ad\u2010Mortimer,\t\r \u00a0J.\t\r \u00a0P.\t\r \u00a0Borg,\t\r \u00a0J.\t\r \u00a0P.\t\r \u00a0Gorvel,\t\r \u00a0S.\t\r \u00a0Meresse,\t\r \u00a0The\t\r \u00a0Salmonella\t\r \u00a0effector\t\r \u00a0protein\t\r \u00a0PipB2\t\r \u00a0is\t\r \u00a0a\t\r \u00a0linker\t\r \u00a0for\t\r \u00a0kinesin-\u00ad\u20101.\t\r \u00a0Proc\t\r \u00a0 Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a0103,\t\r \u00a013497-\u00ad\u201013502\t\r \u00a0(2006).\t\r \u00a0249.\t\r \u00a0 K.\t\r \u00a0Uchiya,\t\r \u00a0M.\t\r \u00a0A.\t\r \u00a0Barbieri,\t\r \u00a0K.\t\r \u00a0Funato,\t\r \u00a0A.\t\r \u00a0H.\t\r \u00a0Shah,\t\r \u00a0P.\t\r \u00a0D.\t\r \u00a0Stahl,\t\r \u00a0E.\t\r \u00a0A.\t\r \u00a0Groisman,\t\r \u00a0A\t\r \u00a0Salmonella\t\r \u00a0virulence\t\r \u00a0protein\t\r \u00a0that\t\r \u00a0inhibits\t\r \u00a0cellular\t\r \u00a0trafficking.\t\r \u00a0EMBO\t\r \u00a0J\t\r \u00a018,\t\r \u00a03924-\u00ad\u20103933\t\r \u00a0(1999).\t\r \u00a0250.\t\r \u00a0 A.\t\r \u00a0H.\t\r \u00a0Lee,\t\r \u00a0M.\t\r \u00a0P.\t\r \u00a0Zareei,\t\r \u00a0S.\t\r \u00a0Daefler,\t\r \u00a0Identification\t\r \u00a0of\t\r \u00a0a\t\r \u00a0NIPSNAP\t\r \u00a0homologue\t\r \u00a0as\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0target\t\r \u00a0for\t\r \u00a0Salmonella\t\r \u00a0virulence\t\r \u00a0protein\t\r \u00a0SpiC.\t\r \u00a0Cell\t\r \u00a0Microbiol\t\r \u00a04,\t\r \u00a0739-\u00ad\u2010750\t\r \u00a0(2002).\t\r \u00a0251.\t\r \u00a0 Y.\t\r \u00a0Shotland,\t\r \u00a0H.\t\r \u00a0Kramer,\t\r \u00a0E.\t\r \u00a0A.\t\r \u00a0Groisman,\t\r \u00a0The\t\r \u00a0Salmonella\t\r \u00a0SpiC\t\r \u00a0protein\t\r \u00a0targets\t\r \u00a0the\t\r \u00a0mammalian\t\r \u00a0Hook3\t\r \u00a0protein\t\r \u00a0function\t\r \u00a0to\t\r \u00a0alter\t\r \u00a0cellular\t\r \u00a0trafficking.\t\r \u00a0Mol\t\r \u00a0 Microbiol\t\r \u00a049,\t\r \u00a01565-\u00ad\u20101576\t\r \u00a0(2003).\t\r \u00a0252.\t\r \u00a0 H.\t\r \u00a0Otto,\t\r \u00a0D.\t\r \u00a0Tezcan-\u00ad\u2010Merdol,\t\r \u00a0R.\t\r \u00a0Girisch,\t\r \u00a0F.\t\r \u00a0Haag,\t\r \u00a0M.\t\r \u00a0Rhen,\t\r \u00a0F.\t\r \u00a0Koch-\u00ad\u2010Nolte,\t\r \u00a0The\t\r \u00a0spvB\t\r \u00a0gene-\u00ad\u2010product\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0virulence\t\r \u00a0plasmid\t\r \u00a0is\t\r \u00a0a\t\r \u00a0mono(ADP-\u00ad\u2010ribosyl)transferase.\t\r \u00a0Mol\t\r \u00a0Microbiol\t\r \u00a037,\t\r \u00a01106-\u00ad\u20101115\t\r \u00a0(2000).\t\r \u00a0253.\t\r \u00a0 D.\t\r \u00a0Tezcan-\u00ad\u2010Merdol,\t\r \u00a0T.\t\r \u00a0Nyman,\t\r \u00a0U.\t\r \u00a0Lindberg,\t\r \u00a0F.\t\r \u00a0Haag,\t\r \u00a0F.\t\r \u00a0Koch-\u00ad\u2010Nolte,\t\r \u00a0M.\t\r \u00a0Rhen,\t\r \u00a0Actin\t\r \u00a0is\t\r \u00a0ADP-\u00ad\u2010ribosylated\t\r \u00a0by\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0virulence-\u00ad\u2010associated\t\r \u00a0protein\t\r \u00a0SpvB.\t\r \u00a0Mol\t\r \u00a0Microbiol\t\r \u00a039,\t\r \u00a0606-\u00ad\u2010619\t\r \u00a0(2001).\t\r \u00a0254.\t\r \u00a0 M.\t\r \u00a0L.\t\r \u00a0Lesnick,\t\r \u00a0N.\t\r \u00a0E.\t\r \u00a0Reiner,\t\r \u00a0J.\t\r \u00a0Fierer,\t\r \u00a0D.\t\r \u00a0G.\t\r \u00a0Guiney,\t\r \u00a0The\t\r \u00a0Salmonella\t\r \u00a0spvB\t\r \u00a0virulence\t\r \u00a0gene\t\r \u00a0encodes\t\r \u00a0an\t\r \u00a0enzyme\t\r \u00a0that\t\r \u00a0ADP-\u00ad\u2010ribosylates\t\r \u00a0actin\t\r \u00a0and\t\r \u00a0destabilizes\t\r \u00a0the\t\r \u00a0cytoskeleton\t\r \u00a0of\t\r \u00a0eukaryotic\t\r \u00a0cells.\t\r \u00a0Mol\t\r \u00a0Microbiol\t\r \u00a039,\t\r \u00a01464-\u00ad\u20101470\t\r \u00a0(2001).\t\r \u00a0255.\t\r \u00a0 E.\t\r \u00a0A.\t\r \u00a0Miao,\t\r \u00a0M.\t\r \u00a0Brittnacher,\t\r \u00a0A.\t\r \u00a0Haraga,\t\r \u00a0R.\t\r \u00a0L.\t\r \u00a0Jeng,\t\r \u00a0M.\t\r \u00a0D.\t\r \u00a0Welch,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0Salmonella\t\r \u00a0effectors\t\r \u00a0translocated\t\r \u00a0across\t\r \u00a0the\t\r \u00a0vacuolar\t\r \u00a0membrane\t\r \u00a0interact\t\r \u00a0with\t\r \u00a0the\t\r \u00a0actin\t\r \u00a0cytoskeleton.\t\r \u00a0Mol\t\r \u00a0Microbiol\t\r \u00a048,\t\r \u00a0401-\u00ad\u2010415\t\r \u00a0(2003).\t\r \u00a0256.\t\r \u00a0 J.\t\r \u00a0Poh,\t\r \u00a0C.\t\r \u00a0Odendall,\t\r \u00a0A.\t\r \u00a0Spanos,\t\r \u00a0C.\t\r \u00a0Boyle,\t\r \u00a0M.\t\r \u00a0Liu,\t\r \u00a0P.\t\r \u00a0Freemont,\t\r \u00a0D.\t\r \u00a0W.\t\r \u00a0Holden,\t\r \u00a0SteC\t\r \u00a0is\t\r \u00a0a\t\r \u00a0Salmonella\t\r \u00a0kinase\t\r \u00a0required\t\r \u00a0for\t\r \u00a0SPI-\u00ad\u20102-\u00ad\u2010dependent\t\r \u00a0F-\u00ad\u2010actin\t\r \u00a0remodelling.\t\r \u00a0Cell\t\r \u00a0 Microbiol\t\r \u00a010,\t\r \u00a020-\u00ad\u201030\t\r \u00a0(2008).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 161\t\r \u00a0 257.\t\r \u00a0 G.\t\r \u00a0Le\t\r \u00a0Negrate,\t\r \u00a0B.\t\r \u00a0Faustin,\t\r \u00a0K.\t\r \u00a0Welsh,\t\r \u00a0M.\t\r \u00a0Loeffler,\t\r \u00a0M.\t\r \u00a0Krajewska,\t\r \u00a0P.\t\r \u00a0Hasegawa,\t\r \u00a0S.\t\r \u00a0Mukherjee,\t\r \u00a0K.\t\r \u00a0Orth,\t\r \u00a0S.\t\r \u00a0Krajewski,\t\r \u00a0A.\t\r \u00a0Godzik,\t\r \u00a0D.\t\r \u00a0G.\t\r \u00a0Guiney,\t\r \u00a0J.\t\r \u00a0C.\t\r \u00a0Reed,\t\r \u00a0Salmonella\t\r \u00a0secreted\t\r \u00a0factor\t\r \u00a0L\t\r \u00a0deubiquitinase\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0inhibits\t\r \u00a0NF-\u00ad\u2010kappaB,\t\r \u00a0suppresses\t\r \u00a0IkappaBalpha\t\r \u00a0ubiquitination\t\r \u00a0and\t\r \u00a0modulates\t\r \u00a0innate\t\r \u00a0immune\t\r \u00a0responses.\t\r \u00a0J\t\r \u00a0Immunol\t\r \u00a0180,\t\r \u00a05045-\u00ad\u20105056\t\r \u00a0(2008).\t\r \u00a0258.\t\r \u00a0 P.\t\r \u00a0Mazurkiewicz,\t\r \u00a0J.\t\r \u00a0Thomas,\t\r \u00a0J.\t\r \u00a0A.\t\r \u00a0Thompson,\t\r \u00a0M.\t\r \u00a0Liu,\t\r \u00a0L.\t\r \u00a0Arbibe,\t\r \u00a0P.\t\r \u00a0Sansonetti,\t\r \u00a0D.\t\r \u00a0W.\t\r \u00a0Holden,\t\r \u00a0SpvC\t\r \u00a0is\t\r \u00a0a\t\r \u00a0Salmonella\t\r \u00a0effector\t\r \u00a0with\t\r \u00a0phosphothreonine\t\r \u00a0lyase\t\r \u00a0activity\t\r \u00a0on\t\r \u00a0host\t\r \u00a0mitogen-\u00ad\u2010activated\t\r \u00a0protein\t\r \u00a0kinases.\t\r \u00a0Mol\t\r \u00a0Microbiol\t\r \u00a067,\t\r \u00a01371-\u00ad\u20101383\t\r \u00a0(2008).\t\r \u00a0259.\t\r \u00a0 P.\t\r \u00a0Nawabi,\t\r \u00a0D.\t\r \u00a0M.\t\r \u00a0Catron,\t\r \u00a0K.\t\r \u00a0Haldar,\t\r \u00a0Esterification\t\r \u00a0of\t\r \u00a0cholesterol\t\r \u00a0by\t\r \u00a0a\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0effector\t\r \u00a0during\t\r \u00a0intracellular\t\r \u00a0Salmonella\t\r \u00a0infection.\t\r \u00a0Mol\t\r \u00a0Microbiol\t\r \u00a068,\t\r \u00a0173-\u00ad\u2010185\t\r \u00a0(2008).\t\r \u00a0260.\t\r \u00a0 B.\t\r \u00a0K.\t\r \u00a0Coombes,\t\r \u00a0M.\t\r \u00a0E.\t\r \u00a0Wickham,\t\r \u00a0N.\t\r \u00a0F.\t\r \u00a0Brown,\t\r \u00a0S.\t\r \u00a0Lemire,\t\r \u00a0L.\t\r \u00a0Bossi,\t\r \u00a0W.\t\r \u00a0W.\t\r \u00a0Hsiao,\t\r \u00a0F.\t\r \u00a0S.\t\r \u00a0Brinkman,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0Genetic\t\r \u00a0and\t\r \u00a0molecular\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0GogB,\t\r \u00a0a\t\r \u00a0phage-\u00ad\u2010encoded\t\r \u00a0type\t\r \u00a0III-\u00ad\u2010secreted\t\r \u00a0substrate\t\r \u00a0in\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0typhimurium\t\r \u00a0with\t\r \u00a0autonomous\t\r \u00a0expression\t\r \u00a0from\t\r \u00a0its\t\r \u00a0associated\t\r \u00a0phage.\t\r \u00a0J\t\r \u00a0Mol\t\r \u00a0 Biol\t\r \u00a0348,\t\r \u00a0817-\u00ad\u2010830\t\r \u00a0(2005).\t\r \u00a0261.\t\r \u00a0 J.\t\r \u00a0A.\t\r \u00a0Freeman,\t\r \u00a0M.\t\r \u00a0E.\t\r \u00a0Ohl,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0The\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0typhimurium\t\r \u00a0translocated\t\r \u00a0effectors\t\r \u00a0SseJ\t\r \u00a0and\t\r \u00a0SifB\t\r \u00a0are\t\r \u00a0targeted\t\r \u00a0to\t\r \u00a0the\t\r \u00a0Salmonella-\u00ad\u2010containing\t\r \u00a0vacuole.\t\r \u00a0Infect\t\r \u00a0Immun\t\r \u00a071,\t\r \u00a0418-\u00ad\u2010427\t\r \u00a0(2003).\t\r \u00a0262.\t\r \u00a0 J.\t\r \u00a0H.\t\r \u00a0Brumell,\t\r \u00a0S.\t\r \u00a0Kujat-\u00ad\u2010Choy,\t\r \u00a0N.\t\r \u00a0F.\t\r \u00a0Brown,\t\r \u00a0B.\t\r \u00a0A.\t\r \u00a0Vallance,\t\r \u00a0L.\t\r \u00a0A.\t\r \u00a0Knodler,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0SopD2\t\r \u00a0is\t\r \u00a0a\t\r \u00a0novel\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secreted\t\r \u00a0effector\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0that\t\r \u00a0targets\t\r \u00a0late\t\r \u00a0endocytic\t\r \u00a0compartments\t\r \u00a0upon\t\r \u00a0delivery\t\r \u00a0into\t\r \u00a0host\t\r \u00a0cells.\t\r \u00a0 Traffic\t\r \u00a04,\t\r \u00a036-\u00ad\u201048\t\r \u00a0(2003).\t\r \u00a0263.\t\r \u00a0 H.\t\r \u00a0Li,\t\r \u00a0H.\t\r \u00a0Xu,\t\r \u00a0Y.\t\r \u00a0Zhou,\t\r \u00a0J.\t\r \u00a0Zhang,\t\r \u00a0C.\t\r \u00a0Long,\t\r \u00a0S.\t\r \u00a0Li,\t\r \u00a0S.\t\r \u00a0Chen,\t\r \u00a0J.\t\r \u00a0M.\t\r \u00a0Zhou,\t\r \u00a0F.\t\r \u00a0Shao,\t\r \u00a0The\t\r \u00a0phosphothreonine\t\r \u00a0lyase\t\r \u00a0activity\t\r \u00a0of\t\r \u00a0a\t\r \u00a0bacterial\t\r \u00a0type\t\r \u00a0III\t\r \u00a0effector\t\r \u00a0family.\t\r \u00a0Science\t\r \u00a0 315,\t\r \u00a01000-\u00ad\u20101003\t\r \u00a0(2007).\t\r \u00a0264.\t\r \u00a0 L.\t\r \u00a0M.\t\r \u00a0McLaughlin,\t\r \u00a0G.\t\r \u00a0R.\t\r \u00a0Govoni,\t\r \u00a0C.\t\r \u00a0Gerke,\t\r \u00a0S.\t\r \u00a0Gopinath,\t\r \u00a0K.\t\r \u00a0Peng,\t\r \u00a0G.\t\r \u00a0Laidlaw,\t\r \u00a0Y.\t\r \u00a0H.\t\r \u00a0Chien,\t\r \u00a0H.\t\r \u00a0W.\t\r \u00a0Jeong,\t\r \u00a0Z.\t\r \u00a0Li,\t\r \u00a0M.\t\r \u00a0D.\t\r \u00a0Brown,\t\r \u00a0D.\t\r \u00a0B.\t\r \u00a0Sacks,\t\r \u00a0D.\t\r \u00a0Monack,\t\r \u00a0The\t\r \u00a0Salmonella\t\r \u00a0SPI2\t\r \u00a0effector\t\r \u00a0SseI\t\r \u00a0mediates\t\r \u00a0long-\u00ad\u2010term\t\r \u00a0systemic\t\r \u00a0infection\t\r \u00a0by\t\r \u00a0modulating\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0migration.\t\r \u00a0PLoS\t\r \u00a0Pathog\t\r \u00a05,\t\r \u00a0e1000671\t\r \u00a0(2009).\t\r \u00a0265.\t\r \u00a0 M.\t\r \u00a0J.\t\r \u00a0Worley,\t\r \u00a0G.\t\r \u00a0S.\t\r \u00a0Nieman,\t\r \u00a0K.\t\r \u00a0Geddes,\t\r \u00a0F.\t\r \u00a0Heffron,\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0disseminates\t\r \u00a0within\t\r \u00a0its\t\r \u00a0host\t\r \u00a0by\t\r \u00a0manipulating\t\r \u00a0the\t\r \u00a0motility\t\r \u00a0of\t\r \u00a0infected\t\r \u00a0cells.\t\r \u00a0 Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a0103,\t\r \u00a017915-\u00ad\u201017920\t\r \u00a0(2006).\t\r \u00a0266.\t\r \u00a0 M.\t\r \u00a0Christen,\t\r \u00a0L.\t\r \u00a0H.\t\r \u00a0Coye,\t\r \u00a0J.\t\r \u00a0S.\t\r \u00a0Hontz,\t\r \u00a0D.\t\r \u00a0L.\t\r \u00a0LaRock,\t\r \u00a0R.\t\r \u00a0A.\t\r \u00a0Pfuetzner,\t\r \u00a0Megha,\t\r \u00a0S.\t\r \u00a0I.\t\r \u00a0Miller,\t\r \u00a0Activation\t\r \u00a0of\t\r \u00a0a\t\r \u00a0bacterial\t\r \u00a0virulence\t\r \u00a0protein\t\r \u00a0by\t\r \u00a0the\t\r \u00a0GTPase\t\r \u00a0RhoA.\t\r \u00a0Sci\t\r \u00a0 Signal\t\r \u00a02,\t\r \u00a0ra71\t\r \u00a0(2009).\t\r \u00a0267.\t\r \u00a0 S.\t\r \u00a0L.\t\r \u00a0Kujat\t\r \u00a0Choy,\t\r \u00a0E.\t\r \u00a0C.\t\r \u00a0Boyle,\t\r \u00a0O.\t\r \u00a0Gal-\u00ad\u2010Mor,\t\r \u00a0D.\t\r \u00a0L.\t\r \u00a0Goode,\t\r \u00a0Y.\t\r \u00a0Valdez,\t\r \u00a0B.\t\r \u00a0A.\t\r \u00a0Vallance,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0SseK1\t\r \u00a0and\t\r \u00a0SseK2\t\r \u00a0are\t\r \u00a0novel\t\r \u00a0translocated\t\r \u00a0proteins\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0typhimurium.\t\r \u00a0Infect\t\r \u00a0Immun\t\r \u00a072,\t\r \u00a05115-\u00ad\u20105125\t\r \u00a0(2004).\t\r \u00a0268.\t\r \u00a0 A.\t\r \u00a0Rytkonen,\t\r \u00a0J.\t\r \u00a0Poh,\t\r \u00a0J.\t\r \u00a0Garmendia,\t\r \u00a0C.\t\r \u00a0Boyle,\t\r \u00a0A.\t\r \u00a0Thompson,\t\r \u00a0M.\t\r \u00a0Liu,\t\r \u00a0P.\t\r \u00a0Freemont,\t\r \u00a0J.\t\r \u00a0C.\t\r \u00a0Hinton,\t\r \u00a0D.\t\r \u00a0W.\t\r \u00a0Holden,\t\r \u00a0SseL,\t\r \u00a0a\t\r \u00a0Salmonella\t\r \u00a0deubiquitinase\t\r \u00a0required\t\r \u00a0for\t\r \u00a0macrophage\t\r \u00a0killing\t\r \u00a0and\t\r \u00a0virulence.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a0104,\t\r \u00a03502-\u00ad\u20103507\t\r \u00a0(2007).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 162\t\r \u00a0 269.\t\r \u00a0 K.\t\r \u00a0Geddes,\t\r \u00a0M.\t\r \u00a0Worley,\t\r \u00a0G.\t\r \u00a0Niemann,\t\r \u00a0F.\t\r \u00a0Heffron,\t\r \u00a0Identification\t\r \u00a0of\t\r \u00a0new\t\r \u00a0secreted\t\r \u00a0effectors\t\r \u00a0in\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0Typhimurium.\t\r \u00a0Infect\t\r \u00a0Immun\t\r \u00a073,\t\r \u00a06260-\u00ad\u20106271\t\r \u00a0(2005).\t\r \u00a0270.\t\r \u00a0 D.\t\r \u00a0V.\t\r \u00a0Zurawski,\t\r \u00a0M.\t\r \u00a0A.\t\r \u00a0Stein,\t\r \u00a0SseA\t\r \u00a0acts\t\r \u00a0as\t\r \u00a0the\t\r \u00a0chaperone\t\r \u00a0for\t\r \u00a0the\t\r \u00a0SseB\t\r \u00a0component\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0Pathogenicity\t\r \u00a0Island\t\r \u00a02\t\r \u00a0translocon.\t\r \u00a0Mol\t\r \u00a0 Microbiol\t\r \u00a047,\t\r \u00a01341-\u00ad\u20101351\t\r \u00a0(2003).\t\r \u00a0271.\t\r \u00a0 J.\t\r \u00a0Ruiz-\u00ad\u2010Albert,\t\r \u00a0R.\t\r \u00a0Mundy,\t\r \u00a0X.\t\r \u00a0J.\t\r \u00a0Yu,\t\r \u00a0C.\t\r \u00a0R.\t\r \u00a0Beuzon,\t\r \u00a0D.\t\r \u00a0W.\t\r \u00a0Holden,\t\r \u00a0SseA\t\r \u00a0is\t\r \u00a0a\t\r \u00a0chaperone\t\r \u00a0for\t\r \u00a0the\t\r \u00a0SseB\t\r \u00a0and\t\r \u00a0SseD\t\r \u00a0translocon\t\r \u00a0components\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0pathogenicity-\u00ad\u2010island-\u00ad\u20102-\u00ad\u2010encoded\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0system.\t\r \u00a0Microbiology\t\r \u00a0149,\t\r \u00a01103-\u00ad\u20101111\t\r \u00a0(2003).\t\r \u00a0272.\t\r \u00a0 D.\t\r \u00a0V.\t\r \u00a0Zurawski,\t\r \u00a0M.\t\r \u00a0A.\t\r \u00a0Stein,\t\r \u00a0The\t\r \u00a0SPI2-\u00ad\u2010encoded\t\r \u00a0SseA\t\r \u00a0chaperone\t\r \u00a0has\t\r \u00a0discrete\t\r \u00a0domains\t\r \u00a0required\t\r \u00a0for\t\r \u00a0SseB\t\r \u00a0stabilization\t\r \u00a0and\t\r \u00a0export,\t\r \u00a0and\t\r \u00a0binds\t\r \u00a0within\t\r \u00a0the\t\r \u00a0C-\u00ad\u2010terminus\t\r \u00a0of\t\r \u00a0SseB\t\r \u00a0and\t\r \u00a0SseD.\t\r \u00a0Microbiology\t\r \u00a0150,\t\r \u00a02055-\u00ad\u20102068\t\r \u00a0(2004).\t\r \u00a0273.\t\r \u00a0 T.\t\r \u00a0Miki,\t\r \u00a0Y.\t\r \u00a0Shibagaki,\t\r \u00a0H.\t\r \u00a0Danbara,\t\r \u00a0N.\t\r \u00a0Okada,\t\r \u00a0Functional\t\r \u00a0characterization\t\r \u00a0of\t\r \u00a0SsaE,\t\r \u00a0a\t\r \u00a0novel\t\r \u00a0chaperone\t\r \u00a0protein\t\r \u00a0of\t\r \u00a0the\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0system\t\r \u00a0encoded\t\r \u00a0by\t\r \u00a0Salmonella\t\r \u00a0pathogenicity\t\r \u00a0island\t\r \u00a02.\t\r \u00a0J\t\r \u00a0Bacteriol\t\r \u00a0191,\t\r \u00a06843-\u00ad\u20106854\t\r \u00a0(2009).\t\r \u00a0274.\t\r \u00a0 C.\t\r \u00a0A.\t\r \u00a0Cooper,\t\r \u00a0K.\t\r \u00a0Zhang,\t\r \u00a0S.\t\r \u00a0N.\t\r \u00a0Andres,\t\r \u00a0Y.\t\r \u00a0Fang,\t\r \u00a0N.\t\r \u00a0A.\t\r \u00a0Kaniuk,\t\r \u00a0M.\t\r \u00a0Hannemann,\t\r \u00a0J.\t\r \u00a0H.\t\r \u00a0Brumell,\t\r \u00a0L.\t\r \u00a0J.\t\r \u00a0Foster,\t\r \u00a0M.\t\r \u00a0S.\t\r \u00a0Junop,\t\r \u00a0B.\t\r \u00a0K.\t\r \u00a0Coombes,\t\r \u00a0Structural\t\r \u00a0and\t\r \u00a0biochemical\t\r \u00a0characterization\t\r \u00a0of\t\r \u00a0SrcA,\t\r \u00a0a\t\r \u00a0multi-\u00ad\u2010cargo\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0chaperone\t\r \u00a0in\t\r \u00a0Salmonella\t\r \u00a0required\t\r \u00a0for\t\r \u00a0pathogenic\t\r \u00a0association\t\r \u00a0with\t\r \u00a0a\t\r \u00a0host.\t\r \u00a0PLoS\t\r \u00a0Pathog\t\r \u00a06,\t\r \u00a0e1000751\t\r \u00a0(2010).\t\r \u00a0275.\t\r \u00a0 M.\t\r \u00a0R.\t\r \u00a0Wilkins,\t\r \u00a0J.\t\r \u00a0C.\t\r \u00a0Sanchez,\t\r \u00a0A.\t\r \u00a0A.\t\r \u00a0Gooley,\t\r \u00a0R.\t\r \u00a0D.\t\r \u00a0Appel,\t\r \u00a0I.\t\r \u00a0Humphery-\u00ad\u2010Smith,\t\r \u00a0D.\t\r \u00a0F.\t\r \u00a0Hochstrasser,\t\r \u00a0K.\t\r \u00a0L.\t\r \u00a0Williams,\t\r \u00a0Progress\t\r \u00a0with\t\r \u00a0proteome\t\r \u00a0projects:\t\r \u00a0why\t\r \u00a0all\t\r \u00a0proteins\t\r \u00a0expressed\t\r \u00a0by\t\r \u00a0a\t\r \u00a0genome\t\r \u00a0should\t\r \u00a0be\t\r \u00a0identified\t\r \u00a0and\t\r \u00a0how\t\r \u00a0to\t\r \u00a0do\t\r \u00a0it.\t\r \u00a0 Biotechnol\t\r \u00a0Genet\t\r \u00a0Eng\t\r \u00a0Rev\t\r \u00a013,\t\r \u00a019-\u00ad\u201050\t\r \u00a0(1996).\t\r \u00a0276.\t\r \u00a0 N.\t\r \u00a0L.\t\r \u00a0Anderson,\t\r \u00a0N.\t\r \u00a0G.\t\r \u00a0Anderson,\t\r \u00a0Proteome\t\r \u00a0and\t\r \u00a0proteomics:\t\r \u00a0new\t\r \u00a0technologies,\t\r \u00a0new\t\r \u00a0concepts,\t\r \u00a0and\t\r \u00a0new\t\r \u00a0words.\t\r \u00a0Electrophoresis\t\r \u00a019,\t\r \u00a01853-\u00ad\u20101861\t\r \u00a0(1998).\t\r \u00a0277.\t\r \u00a0 W.\t\r \u00a0P.\t\r \u00a0Blackstock,\t\r \u00a0M.\t\r \u00a0P.\t\r \u00a0Weir,\t\r \u00a0Proteomics:\t\r \u00a0quantitative\t\r \u00a0and\t\r \u00a0physical\t\r \u00a0mapping\t\r \u00a0of\t\r \u00a0cellular\t\r \u00a0proteins.\t\r \u00a0Trends\t\r \u00a0Biotechnol\t\r \u00a017,\t\r \u00a0121-\u00ad\u2010127\t\r \u00a0(1999).\t\r \u00a0278.\t\r \u00a0 R.\t\r \u00a0H.\t\r \u00a0Aebersold,\t\r \u00a0J.\t\r \u00a0Leavitt,\t\r \u00a0R.\t\r \u00a0A.\t\r \u00a0Saavedra,\t\r \u00a0L.\t\r \u00a0E.\t\r \u00a0Hood,\t\r \u00a0S.\t\r \u00a0B.\t\r \u00a0Kent,\t\r \u00a0Internal\t\r \u00a0amino\t\r \u00a0acid\t\r \u00a0sequence\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0proteins\t\r \u00a0separated\t\r \u00a0by\t\r \u00a0one-\u00ad\u2010\t\r \u00a0or\t\r \u00a0two-\u00ad\u2010dimensional\t\r \u00a0gel\t\r \u00a0electrophoresis\t\r \u00a0after\t\r \u00a0in\t\r \u00a0situ\t\r \u00a0protease\t\r \u00a0digestion\t\r \u00a0on\t\r \u00a0nitrocellulose.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0 Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a084,\t\r \u00a06970-\u00ad\u20106974\t\r \u00a0(1987).\t\r \u00a0279.\t\r \u00a0 P.\t\r \u00a0Tempst,\t\r \u00a0A.\t\r \u00a0J.\t\r \u00a0Link,\t\r \u00a0L.\t\r \u00a0R.\t\r \u00a0Riviere,\t\r \u00a0M.\t\r \u00a0Fleming,\t\r \u00a0C.\t\r \u00a0Elicone,\t\r \u00a0Internal\t\r \u00a0sequence\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0proteins\t\r \u00a0separated\t\r \u00a0on\t\r \u00a0polyacrylamide\t\r \u00a0gels\t\r \u00a0at\t\r \u00a0the\t\r \u00a0submicrogram\t\r \u00a0level:\t\r \u00a0improved\t\r \u00a0methods,\t\r \u00a0applications\t\r \u00a0and\t\r \u00a0gene\t\r \u00a0cloning\t\r \u00a0strategies.\t\r \u00a0 Electrophoresis\t\r \u00a011,\t\r \u00a0537-\u00ad\u2010553\t\r \u00a0(1990).\t\r \u00a0280.\t\r \u00a0 A.\t\r \u00a0Goffeau,\t\r \u00a0B.\t\r \u00a0G.\t\r \u00a0Barrell,\t\r \u00a0H.\t\r \u00a0Bussey,\t\r \u00a0R.\t\r \u00a0W.\t\r \u00a0Davis,\t\r \u00a0B.\t\r \u00a0Dujon,\t\r \u00a0H.\t\r \u00a0Feldmann,\t\r \u00a0F.\t\r \u00a0Galibert,\t\r \u00a0J.\t\r \u00a0D.\t\r \u00a0Hoheisel,\t\r \u00a0C.\t\r \u00a0Jacq,\t\r \u00a0M.\t\r \u00a0Johnston,\t\r \u00a0E.\t\r \u00a0J.\t\r \u00a0Louis,\t\r \u00a0H.\t\r \u00a0W.\t\r \u00a0Mewes,\t\r \u00a0Y.\t\r \u00a0Murakami,\t\r \u00a0P.\t\r \u00a0Philippsen,\t\r \u00a0H.\t\r \u00a0Tettelin,\t\r \u00a0S.\t\r \u00a0G.\t\r \u00a0Oliver,\t\r \u00a0Life\t\r \u00a0with\t\r \u00a06000\t\r \u00a0genes.\t\r \u00a0 Science\t\r \u00a0274,\t\r \u00a0546,\t\r \u00a0563-\u00ad\u2010547\t\r \u00a0(1996).\t\r \u00a0281.\t\r \u00a0 J.\t\r \u00a0C.\t\r \u00a0Venter,\t\r \u00a0M.\t\r \u00a0D.\t\r \u00a0Adams,\t\r \u00a0E.\t\r \u00a0W.\t\r \u00a0Myers,\t\r \u00a0P.\t\r \u00a0W.\t\r \u00a0Li,\t\r \u00a0R.\t\r \u00a0J.\t\r \u00a0Mural,\t\r \u00a0G.\t\r \u00a0G.\t\r \u00a0Sutton,\t\r \u00a0H.\t\r \u00a0O.\t\r \u00a0Smith,\t\r \u00a0M.\t\r \u00a0Yandell,\t\r \u00a0C.\t\r \u00a0A.\t\r \u00a0Evans,\t\r \u00a0R.\t\r \u00a0A.\t\r \u00a0Holt,\t\r \u00a0J.\t\r \u00a0D.\t\r \u00a0Gocayne,\t\r \u00a0P.\t\r \u00a0Amanatides,\t\r \u00a0R.\t\r \u00a0M.\t\r \u00a0Ballew,\t\r \u00a0D.\t\r \u00a0H.\t\r \u00a0Huson,\t\r \u00a0J.\t\r \u00a0R.\t\r \u00a0Wortman,\t\r \u00a0Q.\t\r \u00a0Zhang,\t\r \u00a0C.\t\r \u00a0D.\t\r \u00a0Kodira,\t\r \u00a0X.\t\r \u00a0H.\t\r \u00a0Zheng,\t\r \u00a0L.\t\r \u00a0Chen,\t\r \u00a0M.\t\r \u00a0Skupski,\t\r \u00a0G.\t\r \u00a0Subramanian,\t\r \u00a0P.\t\r \u00a0D.\t\r \u00a0Thomas,\t\r \u00a0J.\t\r \u00a0Zhang,\t\r \u00a0G.\t\r \u00a0L.\t\r \u00a0Gabor\t\r \u00a0Miklos,\t\r \u00a0C.\t\r \u00a0Nelson,\t\r \u00a0S.\t\r \u00a0Broder,\t\r \u00a0A.\t\r \u00a0G.\t\r \u00a0Clark,\t\r \u00a0J.\t\r \u00a0Nadeau,\t\r \u00a0V.\t\r \u00a0A.\t\r \u00a0McKusick,\t\r \u00a0N.\t\r \u00a0Zinder,\t\r \u00a0A.\t\r \u00a0J.\t\r \u00a0 \t\r \u00a0\t\r \u00a0 163\t\r \u00a0 Levine,\t\r \u00a0R.\t\r \u00a0J.\t\r \u00a0Roberts,\t\r \u00a0M.\t\r \u00a0Simon,\t\r \u00a0C.\t\r \u00a0Slayman,\t\r \u00a0M.\t\r \u00a0Hunkapiller,\t\r \u00a0R.\t\r \u00a0Bolanos,\t\r \u00a0A.\t\r \u00a0Delcher,\t\r \u00a0I.\t\r \u00a0Dew,\t\r \u00a0D.\t\r \u00a0Fasulo,\t\r \u00a0M.\t\r \u00a0Flanigan,\t\r \u00a0L.\t\r \u00a0Florea,\t\r \u00a0A.\t\r \u00a0Halpern,\t\r \u00a0S.\t\r \u00a0Hannenhalli,\t\r \u00a0S.\t\r \u00a0Kravitz,\t\r \u00a0S.\t\r \u00a0Levy,\t\r \u00a0C.\t\r \u00a0Mobarry,\t\r \u00a0K.\t\r \u00a0Reinert,\t\r \u00a0K.\t\r \u00a0Remington,\t\r \u00a0J.\t\r \u00a0Abu-\u00ad\u2010Threideh,\t\r \u00a0E.\t\r \u00a0Beasley,\t\r \u00a0K.\t\r \u00a0Biddick,\t\r \u00a0V.\t\r \u00a0Bonazzi,\t\r \u00a0R.\t\r \u00a0Brandon,\t\r \u00a0M.\t\r \u00a0Cargill,\t\r \u00a0I.\t\r \u00a0Chandramouliswaran,\t\r \u00a0R.\t\r \u00a0Charlab,\t\r \u00a0K.\t\r \u00a0Chaturvedi,\t\r \u00a0Z.\t\r \u00a0Deng,\t\r \u00a0V.\t\r \u00a0Di\t\r \u00a0Francesco,\t\r \u00a0P.\t\r \u00a0Dunn,\t\r \u00a0K.\t\r \u00a0Eilbeck,\t\r \u00a0C.\t\r \u00a0Evangelista,\t\r \u00a0A.\t\r \u00a0E.\t\r \u00a0Gabrielian,\t\r \u00a0W.\t\r \u00a0Gan,\t\r \u00a0W.\t\r \u00a0Ge,\t\r \u00a0F.\t\r \u00a0Gong,\t\r \u00a0Z.\t\r \u00a0Gu,\t\r \u00a0P.\t\r \u00a0Guan,\t\r \u00a0T.\t\r \u00a0J.\t\r \u00a0Heiman,\t\r \u00a0M.\t\r \u00a0E.\t\r \u00a0Higgins,\t\r \u00a0R.\t\r \u00a0R.\t\r \u00a0Ji,\t\r \u00a0Z.\t\r \u00a0Ke,\t\r \u00a0K.\t\r \u00a0A.\t\r \u00a0Ketchum,\t\r \u00a0Z.\t\r \u00a0Lai,\t\r \u00a0Y.\t\r \u00a0Lei,\t\r \u00a0Z.\t\r \u00a0Li,\t\r \u00a0J.\t\r \u00a0Li,\t\r \u00a0Y.\t\r \u00a0Liang,\t\r \u00a0X.\t\r \u00a0Lin,\t\r \u00a0F.\t\r \u00a0Lu,\t\r \u00a0G.\t\r \u00a0V.\t\r \u00a0Merkulov,\t\r \u00a0N.\t\r \u00a0Milshina,\t\r \u00a0H.\t\r \u00a0M.\t\r \u00a0Moore,\t\r \u00a0A.\t\r \u00a0K.\t\r \u00a0Naik,\t\r \u00a0V.\t\r \u00a0A.\t\r \u00a0Narayan,\t\r \u00a0B.\t\r \u00a0Neelam,\t\r \u00a0D.\t\r \u00a0Nusskern,\t\r \u00a0D.\t\r \u00a0B.\t\r \u00a0Rusch,\t\r \u00a0S.\t\r \u00a0Salzberg,\t\r \u00a0W.\t\r \u00a0Shao,\t\r \u00a0B.\t\r \u00a0Shue,\t\r \u00a0J.\t\r \u00a0Sun,\t\r \u00a0Z.\t\r \u00a0Wang,\t\r \u00a0A.\t\r \u00a0Wang,\t\r \u00a0X.\t\r \u00a0Wang,\t\r \u00a0J.\t\r \u00a0Wang,\t\r \u00a0M.\t\r \u00a0Wei,\t\r \u00a0R.\t\r \u00a0Wides,\t\r \u00a0C.\t\r \u00a0Xiao,\t\r \u00a0C.\t\r \u00a0Yan,\t\r \u00a0A.\t\r \u00a0Yao,\t\r \u00a0J.\t\r \u00a0Ye,\t\r \u00a0M.\t\r \u00a0Zhan,\t\r \u00a0W.\t\r \u00a0Zhang,\t\r \u00a0H.\t\r \u00a0Zhang,\t\r \u00a0Q.\t\r \u00a0Zhao,\t\r \u00a0L.\t\r \u00a0Zheng,\t\r \u00a0F.\t\r \u00a0Zhong,\t\r \u00a0W.\t\r \u00a0Zhong,\t\r \u00a0S.\t\r \u00a0Zhu,\t\r \u00a0S.\t\r \u00a0Zhao,\t\r \u00a0D.\t\r \u00a0Gilbert,\t\r \u00a0S.\t\r \u00a0Baumhueter,\t\r \u00a0G.\t\r \u00a0Spier,\t\r \u00a0C.\t\r \u00a0Carter,\t\r \u00a0A.\t\r \u00a0Cravchik,\t\r \u00a0T.\t\r \u00a0Woodage,\t\r \u00a0F.\t\r \u00a0Ali,\t\r \u00a0H.\t\r \u00a0An,\t\r \u00a0A.\t\r \u00a0Awe,\t\r \u00a0D.\t\r \u00a0Baldwin,\t\r \u00a0H.\t\r \u00a0Baden,\t\r \u00a0M.\t\r \u00a0Barnstead,\t\r \u00a0I.\t\r \u00a0Barrow,\t\r \u00a0K.\t\r \u00a0Beeson,\t\r \u00a0D.\t\r \u00a0Busam,\t\r \u00a0A.\t\r \u00a0Carver,\t\r \u00a0A.\t\r \u00a0Center,\t\r \u00a0M.\t\r \u00a0L.\t\r \u00a0Cheng,\t\r \u00a0L.\t\r \u00a0Curry,\t\r \u00a0S.\t\r \u00a0Danaher,\t\r \u00a0L.\t\r \u00a0Davenport,\t\r \u00a0R.\t\r \u00a0Desilets,\t\r \u00a0S.\t\r \u00a0Dietz,\t\r \u00a0K.\t\r \u00a0Dodson,\t\r \u00a0L.\t\r \u00a0Doup,\t\r \u00a0S.\t\r \u00a0Ferriera,\t\r \u00a0N.\t\r \u00a0Garg,\t\r \u00a0A.\t\r \u00a0Gluecksmann,\t\r \u00a0B.\t\r \u00a0Hart,\t\r \u00a0J.\t\r \u00a0Haynes,\t\r \u00a0C.\t\r \u00a0Haynes,\t\r \u00a0C.\t\r \u00a0Heiner,\t\r \u00a0S.\t\r \u00a0Hladun,\t\r \u00a0D.\t\r \u00a0Hostin,\t\r \u00a0J.\t\r \u00a0Houck,\t\r \u00a0T.\t\r \u00a0Howland,\t\r \u00a0C.\t\r \u00a0Ibegwam,\t\r \u00a0J.\t\r \u00a0Johnson,\t\r \u00a0F.\t\r \u00a0Kalush,\t\r \u00a0L.\t\r \u00a0Kline,\t\r \u00a0S.\t\r \u00a0Koduru,\t\r \u00a0A.\t\r \u00a0Love,\t\r \u00a0F.\t\r \u00a0Mann,\t\r \u00a0D.\t\r \u00a0May,\t\r \u00a0S.\t\r \u00a0McCawley,\t\r \u00a0T.\t\r \u00a0McIntosh,\t\r \u00a0I.\t\r \u00a0McMullen,\t\r \u00a0M.\t\r \u00a0Moy,\t\r \u00a0L.\t\r \u00a0Moy,\t\r \u00a0B.\t\r \u00a0Murphy,\t\r \u00a0K.\t\r \u00a0Nelson,\t\r \u00a0C.\t\r \u00a0Pfannkoch,\t\r \u00a0E.\t\r \u00a0Pratts,\t\r \u00a0V.\t\r \u00a0Puri,\t\r \u00a0H.\t\r \u00a0Qureshi,\t\r \u00a0M.\t\r \u00a0Reardon,\t\r \u00a0R.\t\r \u00a0Rodriguez,\t\r \u00a0Y.\t\r \u00a0H.\t\r \u00a0Rogers,\t\r \u00a0D.\t\r \u00a0Romblad,\t\r \u00a0B.\t\r \u00a0Ruhfel,\t\r \u00a0R.\t\r \u00a0Scott,\t\r \u00a0C.\t\r \u00a0Sitter,\t\r \u00a0M.\t\r \u00a0Smallwood,\t\r \u00a0E.\t\r \u00a0Stewart,\t\r \u00a0R.\t\r \u00a0Strong,\t\r \u00a0E.\t\r \u00a0Suh,\t\r \u00a0R.\t\r \u00a0Thomas,\t\r \u00a0N.\t\r \u00a0N.\t\r \u00a0Tint,\t\r \u00a0S.\t\r \u00a0Tse,\t\r \u00a0C.\t\r \u00a0Vech,\t\r \u00a0G.\t\r \u00a0Wang,\t\r \u00a0J.\t\r \u00a0Wetter,\t\r \u00a0S.\t\r \u00a0Williams,\t\r \u00a0M.\t\r \u00a0Williams,\t\r \u00a0S.\t\r \u00a0Windsor,\t\r \u00a0E.\t\r \u00a0Winn-\u00ad\u2010Deen,\t\r \u00a0K.\t\r \u00a0Wolfe,\t\r \u00a0J.\t\r \u00a0Zaveri,\t\r \u00a0K.\t\r \u00a0Zaveri,\t\r \u00a0J.\t\r \u00a0F.\t\r \u00a0Abril,\t\r \u00a0R.\t\r \u00a0Guigo,\t\r \u00a0M.\t\r \u00a0J.\t\r \u00a0Campbell,\t\r \u00a0K.\t\r \u00a0V.\t\r \u00a0Sjolander,\t\r \u00a0B.\t\r \u00a0Karlak,\t\r \u00a0A.\t\r \u00a0Kejariwal,\t\r \u00a0H.\t\r \u00a0Mi,\t\r \u00a0B.\t\r \u00a0Lazareva,\t\r \u00a0T.\t\r \u00a0Hatton,\t\r \u00a0A.\t\r \u00a0Narechania,\t\r \u00a0K.\t\r \u00a0Diemer,\t\r \u00a0A.\t\r \u00a0Muruganujan,\t\r \u00a0N.\t\r \u00a0Guo,\t\r \u00a0S.\t\r \u00a0Sato,\t\r \u00a0V.\t\r \u00a0Bafna,\t\r \u00a0S.\t\r \u00a0Istrail,\t\r \u00a0R.\t\r \u00a0Lippert,\t\r \u00a0R.\t\r \u00a0Schwartz,\t\r \u00a0B.\t\r \u00a0Walenz,\t\r \u00a0S.\t\r \u00a0Yooseph,\t\r \u00a0D.\t\r \u00a0Allen,\t\r \u00a0A.\t\r \u00a0Basu,\t\r \u00a0J.\t\r \u00a0Baxendale,\t\r \u00a0L.\t\r \u00a0Blick,\t\r \u00a0M.\t\r \u00a0Caminha,\t\r \u00a0J.\t\r \u00a0Carnes-\u00ad\u2010Stine,\t\r \u00a0P.\t\r \u00a0Caulk,\t\r \u00a0Y.\t\r \u00a0H.\t\r \u00a0Chiang,\t\r \u00a0M.\t\r \u00a0Coyne,\t\r \u00a0C.\t\r \u00a0Dahlke,\t\r \u00a0A.\t\r \u00a0Mays,\t\r \u00a0M.\t\r \u00a0Dombroski,\t\r \u00a0M.\t\r \u00a0Donnelly,\t\r \u00a0D.\t\r \u00a0Ely,\t\r \u00a0S.\t\r \u00a0Esparham,\t\r \u00a0C.\t\r \u00a0Fosler,\t\r \u00a0H.\t\r \u00a0Gire,\t\r \u00a0S.\t\r \u00a0Glanowski,\t\r \u00a0K.\t\r \u00a0Glasser,\t\r \u00a0A.\t\r \u00a0Glodek,\t\r \u00a0M.\t\r \u00a0Gorokhov,\t\r \u00a0K.\t\r \u00a0Graham,\t\r \u00a0B.\t\r \u00a0Gropman,\t\r \u00a0M.\t\r \u00a0Harris,\t\r \u00a0J.\t\r \u00a0Heil,\t\r \u00a0S.\t\r \u00a0Henderson,\t\r \u00a0J.\t\r \u00a0Hoover,\t\r \u00a0D.\t\r \u00a0Jennings,\t\r \u00a0C.\t\r \u00a0Jordan,\t\r \u00a0J.\t\r \u00a0Jordan,\t\r \u00a0J.\t\r \u00a0Kasha,\t\r \u00a0L.\t\r \u00a0Kagan,\t\r \u00a0C.\t\r \u00a0Kraft,\t\r \u00a0A.\t\r \u00a0Levitsky,\t\r \u00a0M.\t\r \u00a0Lewis,\t\r \u00a0X.\t\r \u00a0Liu,\t\r \u00a0J.\t\r \u00a0Lopez,\t\r \u00a0D.\t\r \u00a0Ma,\t\r \u00a0W.\t\r \u00a0Majoros,\t\r \u00a0J.\t\r \u00a0McDaniel,\t\r \u00a0S.\t\r \u00a0Murphy,\t\r \u00a0M.\t\r \u00a0Newman,\t\r \u00a0T.\t\r \u00a0Nguyen,\t\r \u00a0N.\t\r \u00a0Nguyen,\t\r \u00a0M.\t\r \u00a0Nodell,\t\r \u00a0S.\t\r \u00a0Pan,\t\r \u00a0J.\t\r \u00a0Peck,\t\r \u00a0M.\t\r \u00a0Peterson,\t\r \u00a0W.\t\r \u00a0Rowe,\t\r \u00a0R.\t\r \u00a0Sanders,\t\r \u00a0J.\t\r \u00a0Scott,\t\r \u00a0M.\t\r \u00a0Simpson,\t\r \u00a0T.\t\r \u00a0Smith,\t\r \u00a0A.\t\r \u00a0Sprague,\t\r \u00a0T.\t\r \u00a0Stockwell,\t\r \u00a0R.\t\r \u00a0Turner,\t\r \u00a0E.\t\r \u00a0Venter,\t\r \u00a0M.\t\r \u00a0Wang,\t\r \u00a0M.\t\r \u00a0Wen,\t\r \u00a0D.\t\r \u00a0Wu,\t\r \u00a0M.\t\r \u00a0Wu,\t\r \u00a0A.\t\r \u00a0Xia,\t\r \u00a0A.\t\r \u00a0Zandieh,\t\r \u00a0X.\t\r \u00a0Zhu,\t\r \u00a0The\t\r \u00a0sequence\t\r \u00a0of\t\r \u00a0the\t\r \u00a0human\t\r \u00a0genome.\t\r \u00a0Science\t\r \u00a0291,\t\r \u00a01304-\u00ad\u20101351\t\r \u00a0(2001).\t\r \u00a0282.\t\r \u00a0 E.\t\r \u00a0S.\t\r \u00a0Lander,\t\r \u00a0L.\t\r \u00a0M.\t\r \u00a0Linton,\t\r \u00a0B.\t\r \u00a0Birren,\t\r \u00a0C.\t\r \u00a0Nusbaum,\t\r \u00a0M.\t\r \u00a0C.\t\r \u00a0Zody,\t\r \u00a0J.\t\r \u00a0Baldwin,\t\r \u00a0K.\t\r \u00a0Devon,\t\r \u00a0K.\t\r \u00a0Dewar,\t\r \u00a0M.\t\r \u00a0Doyle,\t\r \u00a0W.\t\r \u00a0FitzHugh,\t\r \u00a0R.\t\r \u00a0Funke,\t\r \u00a0D.\t\r \u00a0Gage,\t\r \u00a0K.\t\r \u00a0Harris,\t\r \u00a0A.\t\r \u00a0Heaford,\t\r \u00a0J.\t\r \u00a0Howland,\t\r \u00a0L.\t\r \u00a0Kann,\t\r \u00a0J.\t\r \u00a0Lehoczky,\t\r \u00a0R.\t\r \u00a0LeVine,\t\r \u00a0P.\t\r \u00a0McEwan,\t\r \u00a0K.\t\r \u00a0McKernan,\t\r \u00a0J.\t\r \u00a0Meldrim,\t\r \u00a0J.\t\r \u00a0P.\t\r \u00a0Mesirov,\t\r \u00a0C.\t\r \u00a0Miranda,\t\r \u00a0W.\t\r \u00a0Morris,\t\r \u00a0J.\t\r \u00a0Naylor,\t\r \u00a0C.\t\r \u00a0Raymond,\t\r \u00a0M.\t\r \u00a0Rosetti,\t\r \u00a0R.\t\r \u00a0Santos,\t\r \u00a0A.\t\r \u00a0Sheridan,\t\r \u00a0C.\t\r \u00a0Sougnez,\t\r \u00a0N.\t\r \u00a0Stange-\u00ad\u2010Thomann,\t\r \u00a0N.\t\r \u00a0Stojanovic,\t\r \u00a0A.\t\r \u00a0Subramanian,\t\r \u00a0D.\t\r \u00a0Wyman,\t\r \u00a0J.\t\r \u00a0Rogers,\t\r \u00a0J.\t\r \u00a0Sulston,\t\r \u00a0R.\t\r \u00a0Ainscough,\t\r \u00a0S.\t\r \u00a0Beck,\t\r \u00a0D.\t\r \u00a0Bentley,\t\r \u00a0J.\t\r \u00a0Burton,\t\r \u00a0C.\t\r \u00a0Clee,\t\r \u00a0N.\t\r \u00a0Carter,\t\r \u00a0A.\t\r \u00a0Coulson,\t\r \u00a0R.\t\r \u00a0Deadman,\t\r \u00a0P.\t\r \u00a0Deloukas,\t\r \u00a0A.\t\r \u00a0Dunham,\t\r \u00a0I.\t\r \u00a0Dunham,\t\r \u00a0R.\t\r \u00a0Durbin,\t\r \u00a0L.\t\r \u00a0French,\t\r \u00a0D.\t\r \u00a0Grafham,\t\r \u00a0S.\t\r \u00a0Gregory,\t\r \u00a0T.\t\r \u00a0Hubbard,\t\r \u00a0S.\t\r \u00a0Humphray,\t\r \u00a0A.\t\r \u00a0Hunt,\t\r \u00a0M.\t\r \u00a0Jones,\t\r \u00a0C.\t\r \u00a0Lloyd,\t\r \u00a0A.\t\r \u00a0McMurray,\t\r \u00a0L.\t\r \u00a0Matthews,\t\r \u00a0S.\t\r \u00a0Mercer,\t\r \u00a0S.\t\r \u00a0Milne,\t\r \u00a0J.\t\r \u00a0C.\t\r \u00a0Mullikin,\t\r \u00a0A.\t\r \u00a0Mungall,\t\r \u00a0R.\t\r \u00a0Plumb,\t\r \u00a0M.\t\r \u00a0Ross,\t\r \u00a0R.\t\r \u00a0Shownkeen,\t\r \u00a0S.\t\r \u00a0 \t\r \u00a0\t\r \u00a0 164\t\r \u00a0 Sims,\t\r \u00a0R.\t\r \u00a0H.\t\r \u00a0Waterston,\t\r \u00a0R.\t\r \u00a0K.\t\r \u00a0Wilson,\t\r \u00a0L.\t\r \u00a0W.\t\r \u00a0Hillier,\t\r \u00a0J.\t\r \u00a0D.\t\r \u00a0McPherson,\t\r \u00a0M.\t\r \u00a0A.\t\r \u00a0Marra,\t\r \u00a0E.\t\r \u00a0R.\t\r \u00a0Mardis,\t\r \u00a0L.\t\r \u00a0A.\t\r \u00a0Fulton,\t\r \u00a0A.\t\r \u00a0T.\t\r \u00a0Chinwalla,\t\r \u00a0K.\t\r \u00a0H.\t\r \u00a0Pepin,\t\r \u00a0W.\t\r \u00a0R.\t\r \u00a0Gish,\t\r \u00a0S.\t\r \u00a0L.\t\r \u00a0Chissoe,\t\r \u00a0M.\t\r \u00a0C.\t\r \u00a0Wendl,\t\r \u00a0K.\t\r \u00a0D.\t\r \u00a0Delehaunty,\t\r \u00a0T.\t\r \u00a0L.\t\r \u00a0Miner,\t\r \u00a0A.\t\r \u00a0Delehaunty,\t\r \u00a0J.\t\r \u00a0B.\t\r \u00a0Kramer,\t\r \u00a0L.\t\r \u00a0L.\t\r \u00a0Cook,\t\r \u00a0R.\t\r \u00a0S.\t\r \u00a0Fulton,\t\r \u00a0D.\t\r \u00a0L.\t\r \u00a0Johnson,\t\r \u00a0P.\t\r \u00a0J.\t\r \u00a0Minx,\t\r \u00a0S.\t\r \u00a0W.\t\r \u00a0Clifton,\t\r \u00a0T.\t\r \u00a0Hawkins,\t\r \u00a0E.\t\r \u00a0Branscomb,\t\r \u00a0P.\t\r \u00a0Predki,\t\r \u00a0P.\t\r \u00a0Richardson,\t\r \u00a0S.\t\r \u00a0Wenning,\t\r \u00a0T.\t\r \u00a0Slezak,\t\r \u00a0N.\t\r \u00a0Doggett,\t\r \u00a0J.\t\r \u00a0F.\t\r \u00a0Cheng,\t\r \u00a0A.\t\r \u00a0Olsen,\t\r \u00a0S.\t\r \u00a0Lucas,\t\r \u00a0C.\t\r \u00a0Elkin,\t\r \u00a0E.\t\r \u00a0Uberbacher,\t\r \u00a0M.\t\r \u00a0Frazier,\t\r \u00a0R.\t\r \u00a0A.\t\r \u00a0Gibbs,\t\r \u00a0D.\t\r \u00a0M.\t\r \u00a0Muzny,\t\r \u00a0S.\t\r \u00a0E.\t\r \u00a0Scherer,\t\r \u00a0J.\t\r \u00a0B.\t\r \u00a0Bouck,\t\r \u00a0E.\t\r \u00a0J.\t\r \u00a0Sodergren,\t\r \u00a0K.\t\r \u00a0C.\t\r \u00a0Worley,\t\r \u00a0C.\t\r \u00a0M.\t\r \u00a0Rives,\t\r \u00a0J.\t\r \u00a0H.\t\r \u00a0Gorrell,\t\r \u00a0M.\t\r \u00a0L.\t\r \u00a0Metzker,\t\r \u00a0S.\t\r \u00a0L.\t\r \u00a0Naylor,\t\r \u00a0R.\t\r \u00a0S.\t\r \u00a0Kucherlapati,\t\r \u00a0D.\t\r \u00a0L.\t\r \u00a0Nelson,\t\r \u00a0G.\t\r \u00a0M.\t\r \u00a0Weinstock,\t\r \u00a0Y.\t\r \u00a0Sakaki,\t\r \u00a0A.\t\r \u00a0Fujiyama,\t\r \u00a0M.\t\r \u00a0Hattori,\t\r \u00a0T.\t\r \u00a0Yada,\t\r \u00a0A.\t\r \u00a0Toyoda,\t\r \u00a0T.\t\r \u00a0Itoh,\t\r \u00a0C.\t\r \u00a0Kawagoe,\t\r \u00a0H.\t\r \u00a0Watanabe,\t\r \u00a0Y.\t\r \u00a0Totoki,\t\r \u00a0T.\t\r \u00a0Taylor,\t\r \u00a0J.\t\r \u00a0Weissenbach,\t\r \u00a0R.\t\r \u00a0Heilig,\t\r \u00a0W.\t\r \u00a0Saurin,\t\r \u00a0F.\t\r \u00a0Artiguenave,\t\r \u00a0P.\t\r \u00a0Brottier,\t\r \u00a0T.\t\r \u00a0Bruls,\t\r \u00a0E.\t\r \u00a0Pelletier,\t\r \u00a0C.\t\r \u00a0Robert,\t\r \u00a0P.\t\r \u00a0Wincker,\t\r \u00a0D.\t\r \u00a0R.\t\r \u00a0Smith,\t\r \u00a0L.\t\r \u00a0Doucette-\u00ad\u2010Stamm,\t\r \u00a0M.\t\r \u00a0Rubenfield,\t\r \u00a0K.\t\r \u00a0Weinstock,\t\r \u00a0H.\t\r \u00a0M.\t\r \u00a0Lee,\t\r \u00a0J.\t\r \u00a0Dubois,\t\r \u00a0A.\t\r \u00a0Rosenthal,\t\r \u00a0M.\t\r \u00a0Platzer,\t\r \u00a0G.\t\r \u00a0Nyakatura,\t\r \u00a0S.\t\r \u00a0Taudien,\t\r \u00a0A.\t\r \u00a0Rump,\t\r \u00a0H.\t\r \u00a0Yang,\t\r \u00a0J.\t\r \u00a0Yu,\t\r \u00a0J.\t\r \u00a0Wang,\t\r \u00a0G.\t\r \u00a0Huang,\t\r \u00a0J.\t\r \u00a0Gu,\t\r \u00a0L.\t\r \u00a0Hood,\t\r \u00a0L.\t\r \u00a0Rowen,\t\r \u00a0A.\t\r \u00a0Madan,\t\r \u00a0S.\t\r \u00a0Qin,\t\r \u00a0R.\t\r \u00a0W.\t\r \u00a0Davis,\t\r \u00a0N.\t\r \u00a0A.\t\r \u00a0Federspiel,\t\r \u00a0A.\t\r \u00a0P.\t\r \u00a0Abola,\t\r \u00a0M.\t\r \u00a0J.\t\r \u00a0Proctor,\t\r \u00a0R.\t\r \u00a0M.\t\r \u00a0Myers,\t\r \u00a0J.\t\r \u00a0Schmutz,\t\r \u00a0M.\t\r \u00a0Dickson,\t\r \u00a0J.\t\r \u00a0Grimwood,\t\r \u00a0D.\t\r \u00a0R.\t\r \u00a0Cox,\t\r \u00a0M.\t\r \u00a0V.\t\r \u00a0Olson,\t\r \u00a0R.\t\r \u00a0Kaul,\t\r \u00a0N.\t\r \u00a0Shimizu,\t\r \u00a0K.\t\r \u00a0Kawasaki,\t\r \u00a0S.\t\r \u00a0Minoshima,\t\r \u00a0G.\t\r \u00a0A.\t\r \u00a0Evans,\t\r \u00a0M.\t\r \u00a0Athanasiou,\t\r \u00a0R.\t\r \u00a0Schultz,\t\r \u00a0B.\t\r \u00a0A.\t\r \u00a0Roe,\t\r \u00a0F.\t\r \u00a0Chen,\t\r \u00a0H.\t\r \u00a0Pan,\t\r \u00a0J.\t\r \u00a0Ramser,\t\r \u00a0H.\t\r \u00a0Lehrach,\t\r \u00a0R.\t\r \u00a0Reinhardt,\t\r \u00a0W.\t\r \u00a0R.\t\r \u00a0McCombie,\t\r \u00a0M.\t\r \u00a0de\t\r \u00a0la\t\r \u00a0Bastide,\t\r \u00a0N.\t\r \u00a0Dedhia,\t\r \u00a0H.\t\r \u00a0Blocker,\t\r \u00a0K.\t\r \u00a0Hornischer,\t\r \u00a0G.\t\r \u00a0Nordsiek,\t\r \u00a0R.\t\r \u00a0Agarwala,\t\r \u00a0L.\t\r \u00a0Aravind,\t\r \u00a0J.\t\r \u00a0A.\t\r \u00a0Bailey,\t\r \u00a0A.\t\r \u00a0Bateman,\t\r \u00a0S.\t\r \u00a0Batzoglou,\t\r \u00a0E.\t\r \u00a0Birney,\t\r \u00a0P.\t\r \u00a0Bork,\t\r \u00a0D.\t\r \u00a0G.\t\r \u00a0Brown,\t\r \u00a0C.\t\r \u00a0B.\t\r \u00a0Burge,\t\r \u00a0L.\t\r \u00a0Cerutti,\t\r \u00a0H.\t\r \u00a0C.\t\r \u00a0Chen,\t\r \u00a0D.\t\r \u00a0Church,\t\r \u00a0M.\t\r \u00a0Clamp,\t\r \u00a0R.\t\r \u00a0R.\t\r \u00a0Copley,\t\r \u00a0T.\t\r \u00a0Doerks,\t\r \u00a0S.\t\r \u00a0R.\t\r \u00a0Eddy,\t\r \u00a0E.\t\r \u00a0E.\t\r \u00a0Eichler,\t\r \u00a0T.\t\r \u00a0S.\t\r \u00a0Furey,\t\r \u00a0J.\t\r \u00a0Galagan,\t\r \u00a0J.\t\r \u00a0G.\t\r \u00a0Gilbert,\t\r \u00a0C.\t\r \u00a0Harmon,\t\r \u00a0Y.\t\r \u00a0Hayashizaki,\t\r \u00a0D.\t\r \u00a0Haussler,\t\r \u00a0H.\t\r \u00a0Hermjakob,\t\r \u00a0K.\t\r \u00a0Hokamp,\t\r \u00a0W.\t\r \u00a0Jang,\t\r \u00a0L.\t\r \u00a0S.\t\r \u00a0Johnson,\t\r \u00a0T.\t\r \u00a0A.\t\r \u00a0Jones,\t\r \u00a0S.\t\r \u00a0Kasif,\t\r \u00a0A.\t\r \u00a0Kaspryzk,\t\r \u00a0S.\t\r \u00a0Kennedy,\t\r \u00a0W.\t\r \u00a0J.\t\r \u00a0Kent,\t\r \u00a0P.\t\r \u00a0Kitts,\t\r \u00a0E.\t\r \u00a0V.\t\r \u00a0Koonin,\t\r \u00a0I.\t\r \u00a0Korf,\t\r \u00a0D.\t\r \u00a0Kulp,\t\r \u00a0D.\t\r \u00a0Lancet,\t\r \u00a0T.\t\r \u00a0M.\t\r \u00a0Lowe,\t\r \u00a0A.\t\r \u00a0McLysaght,\t\r \u00a0T.\t\r \u00a0Mikkelsen,\t\r \u00a0J.\t\r \u00a0V.\t\r \u00a0Moran,\t\r \u00a0N.\t\r \u00a0Mulder,\t\r \u00a0V.\t\r \u00a0J.\t\r \u00a0Pollara,\t\r \u00a0C.\t\r \u00a0P.\t\r \u00a0Ponting,\t\r \u00a0G.\t\r \u00a0Schuler,\t\r \u00a0J.\t\r \u00a0Schultz,\t\r \u00a0G.\t\r \u00a0Slater,\t\r \u00a0A.\t\r \u00a0F.\t\r \u00a0Smit,\t\r \u00a0E.\t\r \u00a0Stupka,\t\r \u00a0J.\t\r \u00a0Szustakowski,\t\r \u00a0D.\t\r \u00a0Thierry-\u00ad\u2010Mieg,\t\r \u00a0J.\t\r \u00a0Thierry-\u00ad\u2010Mieg,\t\r \u00a0L.\t\r \u00a0Wagner,\t\r \u00a0J.\t\r \u00a0Wallis,\t\r \u00a0R.\t\r \u00a0Wheeler,\t\r \u00a0A.\t\r \u00a0Williams,\t\r \u00a0Y.\t\r \u00a0I.\t\r \u00a0Wolf,\t\r \u00a0K.\t\r \u00a0H.\t\r \u00a0Wolfe,\t\r \u00a0S.\t\r \u00a0P.\t\r \u00a0Yang,\t\r \u00a0R.\t\r \u00a0F.\t\r \u00a0Yeh,\t\r \u00a0F.\t\r \u00a0Collins,\t\r \u00a0M.\t\r \u00a0S.\t\r \u00a0Guyer,\t\r \u00a0J.\t\r \u00a0Peterson,\t\r \u00a0A.\t\r \u00a0Felsenfeld,\t\r \u00a0K.\t\r \u00a0A.\t\r \u00a0Wetterstrand,\t\r \u00a0A.\t\r \u00a0Patrinos,\t\r \u00a0M.\t\r \u00a0J.\t\r \u00a0Morgan,\t\r \u00a0P.\t\r \u00a0de\t\r \u00a0Jong,\t\r \u00a0J.\t\r \u00a0J.\t\r \u00a0Catanese,\t\r \u00a0K.\t\r \u00a0Osoegawa,\t\r \u00a0H.\t\r \u00a0Shizuya,\t\r \u00a0S.\t\r \u00a0Choi,\t\r \u00a0Y.\t\r \u00a0J.\t\r \u00a0Chen,\t\r \u00a0Initial\t\r \u00a0sequencing\t\r \u00a0and\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0the\t\r \u00a0human\t\r \u00a0genome.\t\r \u00a0Nature\t\r \u00a0409,\t\r \u00a0860-\u00ad\u2010921\t\r \u00a0(2001).\t\r \u00a0283.\t\r \u00a0 W.\t\r \u00a0J.\t\r \u00a0Henzel,\t\r \u00a0T.\t\r \u00a0M.\t\r \u00a0Billeci,\t\r \u00a0J.\t\r \u00a0T.\t\r \u00a0Stults,\t\r \u00a0S.\t\r \u00a0C.\t\r \u00a0Wong,\t\r \u00a0C.\t\r \u00a0Grimley,\t\r \u00a0C.\t\r \u00a0Watanabe,\t\r \u00a0Identifying\t\r \u00a0proteins\t\r \u00a0from\t\r \u00a0two-\u00ad\u2010dimensional\t\r \u00a0gels\t\r \u00a0by\t\r \u00a0molecular\t\r \u00a0mass\t\r \u00a0searching\t\r \u00a0of\t\r \u00a0peptide\t\r \u00a0fragments\t\r \u00a0in\t\r \u00a0protein\t\r \u00a0sequence\t\r \u00a0databases.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a0 90,\t\r \u00a05011-\u00ad\u20105015\t\r \u00a0(1993).\t\r \u00a0284.\t\r \u00a0 M.\t\r \u00a0Mann,\t\r \u00a0P.\t\r \u00a0Hojrup,\t\r \u00a0P.\t\r \u00a0Roepstorff,\t\r \u00a0Use\t\r \u00a0of\t\r \u00a0mass\t\r \u00a0spectrometric\t\r \u00a0molecular\t\r \u00a0weight\t\r \u00a0information\t\r \u00a0to\t\r \u00a0identify\t\r \u00a0proteins\t\r \u00a0in\t\r \u00a0sequence\t\r \u00a0databases.\t\r \u00a0Biol\t\r \u00a0Mass\t\r \u00a0 Spectrom\t\r \u00a022,\t\r \u00a0338-\u00ad\u2010345\t\r \u00a0(1993).\t\r \u00a0285.\t\r \u00a0 P.\t\r \u00a0H.\t\r \u00a0O'Farrell,\t\r \u00a0High\t\r \u00a0resolution\t\r \u00a0two-\u00ad\u2010dimensional\t\r \u00a0electrophoresis\t\r \u00a0of\t\r \u00a0proteins.\t\r \u00a0J\t\r \u00a0 Biol\t\r \u00a0Chem\t\r \u00a0250,\t\r \u00a04007-\u00ad\u20104021\t\r \u00a0(1975).\t\r \u00a0286.\t\r \u00a0 N.\t\r \u00a0L.\t\r \u00a0Anderson,\t\r \u00a0A.\t\r \u00a0D.\t\r \u00a0Matheson,\t\r \u00a0S.\t\r \u00a0Steiner,\t\r \u00a0Proteomics:\t\r \u00a0applications\t\r \u00a0in\t\r \u00a0basic\t\r \u00a0and\t\r \u00a0applied\t\r \u00a0biology.\t\r \u00a0Curr\t\r \u00a0Opin\t\r \u00a0Biotechnol\t\r \u00a011,\t\r \u00a0408-\u00ad\u2010412\t\r \u00a0(2000).\t\r \u00a0287.\t\r \u00a0 S.\t\r \u00a0P.\t\r \u00a0Gygi,\t\r \u00a0G.\t\r \u00a0L.\t\r \u00a0Corthals,\t\r \u00a0Y.\t\r \u00a0Zhang,\t\r \u00a0Y.\t\r \u00a0Rochon,\t\r \u00a0R.\t\r \u00a0Aebersold,\t\r \u00a0Evaluation\t\r \u00a0of\t\r \u00a0two-\u00ad\u2010dimensional\t\r \u00a0gel\t\r \u00a0electrophoresis-\u00ad\u2010based\t\r \u00a0proteome\t\r \u00a0analysis\t\r \u00a0technology.\t\r \u00a0Proc\t\r \u00a0 Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a097,\t\r \u00a09390-\u00ad\u20109395\t\r \u00a0(2000).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 165\t\r \u00a0 288.\t\r \u00a0 S.\t\r \u00a0D.\t\r \u00a0Patterson,\t\r \u00a0R.\t\r \u00a0Aebersold,\t\r \u00a0Mass\t\r \u00a0spectrometric\t\r \u00a0approaches\t\r \u00a0for\t\r \u00a0the\t\r \u00a0identification\t\r \u00a0of\t\r \u00a0gel-\u00ad\u2010separated\t\r \u00a0proteins.\t\r \u00a0Electrophoresis\t\r \u00a016,\t\r \u00a01791-\u00ad\u20101814\t\r \u00a0(1995).\t\r \u00a0289.\t\r \u00a0 R.\t\r \u00a0E.\t\r \u00a0Jenkins,\t\r \u00a0S.\t\r \u00a0R.\t\r \u00a0Pennington,\t\r \u00a0Arrays\t\r \u00a0for\t\r \u00a0protein\t\r \u00a0expression\t\r \u00a0profiling:\t\r \u00a0towards\t\r \u00a0a\t\r \u00a0viable\t\r \u00a0alternative\t\r \u00a0to\t\r \u00a0two-\u00ad\u2010dimensional\t\r \u00a0gel\t\r \u00a0electrophoresis?\t\r \u00a0 Proteomics\t\r \u00a01,\t\r \u00a013-\u00ad\u201029\t\r \u00a0(2001).\t\r \u00a0290.\t\r \u00a0 S.\t\r \u00a0Pelech,\t\r \u00a0L.\t\r \u00a0Jelinkova,\t\r \u00a0A.\t\r \u00a0Susor,\t\r \u00a0H.\t\r \u00a0Zhang,\t\r \u00a0X.\t\r \u00a0Shi,\t\r \u00a0A.\t\r \u00a0Pavlok,\t\r \u00a0M.\t\r \u00a0Kubelka,\t\r \u00a0H.\t\r \u00a0Kovarova,\t\r \u00a0Antibody\t\r \u00a0microarray\t\r \u00a0analyses\t\r \u00a0of\t\r \u00a0signal\t\r \u00a0transduction\t\r \u00a0protein\t\r \u00a0expression\t\r \u00a0and\t\r \u00a0phosphorylation\t\r \u00a0during\t\r \u00a0porcine\t\r \u00a0oocyte\t\r \u00a0maturation.\t\r \u00a0J\t\r \u00a0 Proteome\t\r \u00a0Res\t\r \u00a07,\t\r \u00a02860-\u00ad\u20102871\t\r \u00a0(2008).\t\r \u00a0291.\t\r \u00a0 P.\t\r \u00a0Mitchell,\t\r \u00a0A\t\r \u00a0perspective\t\r \u00a0on\t\r \u00a0protein\t\r \u00a0microarrays.\t\r \u00a0Nat\t\r \u00a0Biotechnol\t\r \u00a020,\t\r \u00a0225-\u00ad\u2010229\t\r \u00a0(2002).\t\r \u00a0292.\t\r \u00a0 A.\t\r \u00a0Armirotti,\t\r \u00a0G.\t\r \u00a0Damonte,\t\r \u00a0Achievements\t\r \u00a0and\t\r \u00a0perspectives\t\r \u00a0of\t\r \u00a0top-\u00ad\u2010down\t\r \u00a0proteomics.\t\r \u00a0Proteomics\t\r \u00a010,\t\r \u00a03566-\u00ad\u20103576\t\r \u00a0(2010).\t\r \u00a0293.\t\r \u00a0 D.\t\r \u00a0F.\t\r \u00a0Hunt,\t\r \u00a0R.\t\r \u00a0A.\t\r \u00a0Henderson,\t\r \u00a0J.\t\r \u00a0Shabanowitz,\t\r \u00a0K.\t\r \u00a0Sakaguchi,\t\r \u00a0H.\t\r \u00a0Michel,\t\r \u00a0N.\t\r \u00a0Sevilir,\t\r \u00a0A.\t\r \u00a0L.\t\r \u00a0Cox,\t\r \u00a0E.\t\r \u00a0Appella,\t\r \u00a0V.\t\r \u00a0H.\t\r \u00a0Engelhard,\t\r \u00a0Characterization\t\r \u00a0of\t\r \u00a0peptides\t\r \u00a0bound\t\r \u00a0to\t\r \u00a0the\t\r \u00a0class\t\r \u00a0I\t\r \u00a0MHC\t\r \u00a0molecule\t\r \u00a0HLA-\u00ad\u2010A2.1\t\r \u00a0by\t\r \u00a0mass\t\r \u00a0spectrometry.\t\r \u00a0Science\t\r \u00a0255,\t\r \u00a01261-\u00ad\u20101263\t\r \u00a0(1992).\t\r \u00a0294.\t\r \u00a0 R.\t\r \u00a0Aebersold,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Mass\t\r \u00a0spectrometry-\u00ad\u2010based\t\r \u00a0proteomics.\t\r \u00a0Nature\t\r \u00a0422,\t\r \u00a0198-\u00ad\u2010207\t\r \u00a0(2003).\t\r \u00a0295.\t\r \u00a0 A.\t\r \u00a0C.\t\r \u00a0Gavin,\t\r \u00a0M.\t\r \u00a0Bosche,\t\r \u00a0R.\t\r \u00a0Krause,\t\r \u00a0P.\t\r \u00a0Grandi,\t\r \u00a0M.\t\r \u00a0Marzioch,\t\r \u00a0A.\t\r \u00a0Bauer,\t\r \u00a0J.\t\r \u00a0Schultz,\t\r \u00a0J.\t\r \u00a0M.\t\r \u00a0Rick,\t\r \u00a0A.\t\r \u00a0M.\t\r \u00a0Michon,\t\r \u00a0C.\t\r \u00a0M.\t\r \u00a0Cruciat,\t\r \u00a0M.\t\r \u00a0Remor,\t\r \u00a0C.\t\r \u00a0Hofert,\t\r \u00a0M.\t\r \u00a0Schelder,\t\r \u00a0M.\t\r \u00a0Brajenovic,\t\r \u00a0H.\t\r \u00a0Ruffner,\t\r \u00a0A.\t\r \u00a0Merino,\t\r \u00a0K.\t\r \u00a0Klein,\t\r \u00a0M.\t\r \u00a0Hudak,\t\r \u00a0D.\t\r \u00a0Dickson,\t\r \u00a0T.\t\r \u00a0Rudi,\t\r \u00a0V.\t\r \u00a0Gnau,\t\r \u00a0A.\t\r \u00a0Bauch,\t\r \u00a0S.\t\r \u00a0Bastuck,\t\r \u00a0B.\t\r \u00a0Huhse,\t\r \u00a0C.\t\r \u00a0Leutwein,\t\r \u00a0M.\t\r \u00a0A.\t\r \u00a0Heurtier,\t\r \u00a0R.\t\r \u00a0R.\t\r \u00a0Copley,\t\r \u00a0A.\t\r \u00a0Edelmann,\t\r \u00a0E.\t\r \u00a0Querfurth,\t\r \u00a0V.\t\r \u00a0Rybin,\t\r \u00a0G.\t\r \u00a0Drewes,\t\r \u00a0M.\t\r \u00a0Raida,\t\r \u00a0T.\t\r \u00a0Bouwmeester,\t\r \u00a0P.\t\r \u00a0Bork,\t\r \u00a0B.\t\r \u00a0Seraphin,\t\r \u00a0B.\t\r \u00a0Kuster,\t\r \u00a0G.\t\r \u00a0Neubauer,\t\r \u00a0G.\t\r \u00a0Superti-\u00ad\u2010Furga,\t\r \u00a0Functional\t\r \u00a0organization\t\r \u00a0of\t\r \u00a0the\t\r \u00a0yeast\t\r \u00a0proteome\t\r \u00a0by\t\r \u00a0systematic\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0protein\t\r \u00a0complexes.\t\r \u00a0Nature\t\r \u00a0415,\t\r \u00a0141-\u00ad\u2010147\t\r \u00a0(2002).\t\r \u00a0296.\t\r \u00a0 L.\t\r \u00a0J.\t\r \u00a0Foster,\t\r \u00a0C.\t\r \u00a0L.\t\r \u00a0de\t\r \u00a0Hoog,\t\r \u00a0Y.\t\r \u00a0Zhang,\t\r \u00a0X.\t\r \u00a0Xie,\t\r \u00a0V.\t\r \u00a0K.\t\r \u00a0Mootha,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0A\t\r \u00a0mammalian\t\r \u00a0organelle\t\r \u00a0map\t\r \u00a0by\t\r \u00a0protein\t\r \u00a0correlation\t\r \u00a0profiling.\t\r \u00a0Cell\t\r \u00a0125,\t\r \u00a0187-\u00ad\u2010199\t\r \u00a0(2006).\t\r \u00a0297.\t\r \u00a0 J.\t\r \u00a0V.\t\r \u00a0Olsen,\t\r \u00a0B.\t\r \u00a0Blagoev,\t\r \u00a0F.\t\r \u00a0Gnad,\t\r \u00a0B.\t\r \u00a0Macek,\t\r \u00a0C.\t\r \u00a0Kumar,\t\r \u00a0P.\t\r \u00a0Mortensen,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Global,\t\r \u00a0in\t\r \u00a0vivo,\t\r \u00a0and\t\r \u00a0site-\u00ad\u2010specific\t\r \u00a0phosphorylation\t\r \u00a0dynamics\t\r \u00a0in\t\r \u00a0signaling\t\r \u00a0networks.\t\r \u00a0Cell\t\r \u00a0127,\t\r \u00a0635-\u00ad\u2010648\t\r \u00a0(2006).\t\r \u00a0298.\t\r \u00a0 P.\t\r \u00a0Picotti,\t\r \u00a0B.\t\r \u00a0Bodenmiller,\t\r \u00a0L.\t\r \u00a0N.\t\r \u00a0Mueller,\t\r \u00a0B.\t\r \u00a0Domon,\t\r \u00a0R.\t\r \u00a0Aebersold,\t\r \u00a0Full\t\r \u00a0dynamic\t\r \u00a0range\t\r \u00a0proteome\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0S.\t\r \u00a0cerevisiae\t\r \u00a0by\t\r \u00a0targeted\t\r \u00a0proteomics.\t\r \u00a0Cell\t\r \u00a0138,\t\r \u00a0795-\u00ad\u2010806\t\r \u00a0(2009).\t\r \u00a0299.\t\r \u00a0 H.\t\r \u00a0R.\t\r \u00a0Morris,\t\r \u00a0T.\t\r \u00a0Paxton,\t\r \u00a0A.\t\r \u00a0Dell,\t\r \u00a0J.\t\r \u00a0Langhorne,\t\r \u00a0M.\t\r \u00a0Berg,\t\r \u00a0R.\t\r \u00a0S.\t\r \u00a0Bordoli,\t\r \u00a0J.\t\r \u00a0Hoyes,\t\r \u00a0R.\t\r \u00a0H.\t\r \u00a0Bateman,\t\r \u00a0High\t\r \u00a0sensitivity\t\r \u00a0collisionally-\u00ad\u2010activated\t\r \u00a0decomposition\t\r \u00a0tandem\t\r \u00a0mass\t\r \u00a0spectrometry\t\r \u00a0on\t\r \u00a0a\t\r \u00a0novel\t\r \u00a0quadrupole\/orthogonal-\u00ad\u2010acceleration\t\r \u00a0time-\u00ad\u2010of-\u00ad\u2010flight\t\r \u00a0mass\t\r \u00a0spectrometer.\t\r \u00a0Rapid\t\r \u00a0Commun\t\r \u00a0Mass\t\r \u00a0Spectrom\t\r \u00a010,\t\r \u00a0889-\u00ad\u2010896\t\r \u00a0(1996).\t\r \u00a0300.\t\r \u00a0 J.\t\r \u00a0C.\t\r \u00a0Schwartz,\t\r \u00a0M.\t\r \u00a0W.\t\r \u00a0Senko,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Syka,\t\r \u00a0A\t\r \u00a0two-\u00ad\u2010dimensional\t\r \u00a0quadrupole\t\r \u00a0ion\t\r \u00a0trap\t\r \u00a0mass\t\r \u00a0spectrometer.\t\r \u00a0J\t\r \u00a0Am\t\r \u00a0Soc\t\r \u00a0Mass\t\r \u00a0Spectrom\t\r \u00a013,\t\r \u00a0659-\u00ad\u2010669\t\r \u00a0(2002).\t\r \u00a0301.\t\r \u00a0 Q.\t\r \u00a0Hu,\t\r \u00a0R.\t\r \u00a0J.\t\r \u00a0Noll,\t\r \u00a0H.\t\r \u00a0Li,\t\r \u00a0A.\t\r \u00a0Makarov,\t\r \u00a0M.\t\r \u00a0Hardman,\t\r \u00a0R.\t\r \u00a0Graham\t\r \u00a0Cooks,\t\r \u00a0The\t\r \u00a0Orbitrap:\t\r \u00a0a\t\r \u00a0new\t\r \u00a0mass\t\r \u00a0spectrometer.\t\r \u00a0J\t\r \u00a0Mass\t\r \u00a0Spectrom\t\r \u00a040,\t\r \u00a0430-\u00ad\u2010443\t\r \u00a0(2005).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 166\t\r \u00a0 302.\t\r \u00a0 T.\t\r \u00a0L.\t\r \u00a0Quenzer,\t\r \u00a0M.\t\r \u00a0R.\t\r \u00a0Emmett,\t\r \u00a0C.\t\r \u00a0L.\t\r \u00a0Hendrickson,\t\r \u00a0P.\t\r \u00a0H.\t\r \u00a0Kelly,\t\r \u00a0A.\t\r \u00a0G.\t\r \u00a0Marshall,\t\r \u00a0High\t\r \u00a0sensitivity\t\r \u00a0Fourier\t\r \u00a0transform\t\r \u00a0ion\t\r \u00a0cyclotron\t\r \u00a0resonance\t\r \u00a0mass\t\r \u00a0spectrometry\t\r \u00a0for\t\r \u00a0biological\t\r \u00a0analysis\t\r \u00a0with\t\r \u00a0nano-\u00ad\u2010LC\t\r \u00a0and\t\r \u00a0microelectrospray\t\r \u00a0ionization.\t\r \u00a0Anal\t\r \u00a0 Chem\t\r \u00a073,\t\r \u00a01721-\u00ad\u20101725\t\r \u00a0(2001).\t\r \u00a0303.\t\r \u00a0 A.\t\r \u00a0Makarov,\t\r \u00a0E.\t\r \u00a0Denisov,\t\r \u00a0A.\t\r \u00a0Kholomeev,\t\r \u00a0W.\t\r \u00a0Balschun,\t\r \u00a0O.\t\r \u00a0Lange,\t\r \u00a0K.\t\r \u00a0Strupat,\t\r \u00a0S.\t\r \u00a0Horning,\t\r \u00a0Performance\t\r \u00a0evaluation\t\r \u00a0of\t\r \u00a0a\t\r \u00a0hybrid\t\r \u00a0linear\t\r \u00a0ion\t\r \u00a0trap\/orbitrap\t\r \u00a0mass\t\r \u00a0spectrometer.\t\r \u00a0Anal\t\r \u00a0Chem\t\r \u00a078,\t\r \u00a02113-\u00ad\u20102120\t\r \u00a0(2006).\t\r \u00a0304.\t\r \u00a0 Q.\t\r \u00a0W.\t\r \u00a0Chan,\t\r \u00a0C.\t\r \u00a0G.\t\r \u00a0Howes,\t\r \u00a0L.\t\r \u00a0J.\t\r \u00a0Foster,\t\r \u00a0Quantitative\t\r \u00a0comparison\t\r \u00a0of\t\r \u00a0caste\t\r \u00a0differences\t\r \u00a0in\t\r \u00a0honeybee\t\r \u00a0hemolymph.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Proteomics\t\r \u00a05,\t\r \u00a02252-\u00ad\u20102262\t\r \u00a0(2006).\t\r \u00a0305.\t\r \u00a0 J.\t\r \u00a0V.\t\r \u00a0Olsen,\t\r \u00a0L.\t\r \u00a0M.\t\r \u00a0de\t\r \u00a0Godoy,\t\r \u00a0G.\t\r \u00a0Li,\t\r \u00a0B.\t\r \u00a0Macek,\t\r \u00a0P.\t\r \u00a0Mortensen,\t\r \u00a0R.\t\r \u00a0Pesch,\t\r \u00a0A.\t\r \u00a0Makarov,\t\r \u00a0O.\t\r \u00a0Lange,\t\r \u00a0S.\t\r \u00a0Horning,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Parts\t\r \u00a0per\t\r \u00a0million\t\r \u00a0mass\t\r \u00a0accuracy\t\r \u00a0on\t\r \u00a0an\t\r \u00a0Orbitrap\t\r \u00a0mass\t\r \u00a0spectrometer\t\r \u00a0via\t\r \u00a0lock\t\r \u00a0mass\t\r \u00a0injection\t\r \u00a0into\t\r \u00a0a\t\r \u00a0C-\u00ad\u2010trap.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0 Proteomics\t\r \u00a04,\t\r \u00a02010-\u00ad\u20102021\t\r \u00a0(2005).\t\r \u00a0306.\t\r \u00a0 G.\t\r \u00a0C.\t\r \u00a0McAlister,\t\r \u00a0W.\t\r \u00a0T.\t\r \u00a0Berggren,\t\r \u00a0J.\t\r \u00a0Griep-\u00ad\u2010Raming,\t\r \u00a0S.\t\r \u00a0Horning,\t\r \u00a0A.\t\r \u00a0Makarov,\t\r \u00a0D.\t\r \u00a0Phanstiel,\t\r \u00a0G.\t\r \u00a0Stafford,\t\r \u00a0D.\t\r \u00a0L.\t\r \u00a0Swaney,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Syka,\t\r \u00a0V.\t\r \u00a0Zabrouskov,\t\r \u00a0J.\t\r \u00a0J.\t\r \u00a0Coon,\t\r \u00a0A\t\r \u00a0proteomics\t\r \u00a0grade\t\r \u00a0electron\t\r \u00a0transfer\t\r \u00a0dissociation-\u00ad\u2010enabled\t\r \u00a0hybrid\t\r \u00a0linear\t\r \u00a0ion\t\r \u00a0trap-\u00ad\u2010orbitrap\t\r \u00a0mass\t\r \u00a0spectrometer.\t\r \u00a0J\t\r \u00a0Proteome\t\r \u00a0Res\t\r \u00a07,\t\r \u00a03127-\u00ad\u20103136\t\r \u00a0(2008).\t\r \u00a0307.\t\r \u00a0 J.\t\r \u00a0V.\t\r \u00a0Olsen,\t\r \u00a0B.\t\r \u00a0Macek,\t\r \u00a0O.\t\r \u00a0Lange,\t\r \u00a0A.\t\r \u00a0Makarov,\t\r \u00a0S.\t\r \u00a0Horning,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Higher-\u00ad\u2010energy\t\r \u00a0C-\u00ad\u2010trap\t\r \u00a0dissociation\t\r \u00a0for\t\r \u00a0peptide\t\r \u00a0modification\t\r \u00a0analysis.\t\r \u00a0Nat\t\r \u00a0Methods\t\r \u00a04,\t\r \u00a0709-\u00ad\u2010712\t\r \u00a0(2007).\t\r \u00a0308.\t\r \u00a0 R.\t\r \u00a0S.\t\r \u00a0Johnson,\t\r \u00a0J.\t\r \u00a0A.\t\r \u00a0Taylor,\t\r \u00a0Searching\t\r \u00a0sequence\t\r \u00a0databases\t\r \u00a0via\t\r \u00a0de\t\r \u00a0novo\t\r \u00a0peptide\t\r \u00a0sequencing\t\r \u00a0by\t\r \u00a0tandem\t\r \u00a0mass\t\r \u00a0spectrometry.\t\r \u00a0Mol\t\r \u00a0Biotechnol\t\r \u00a022,\t\r \u00a0301-\u00ad\u2010315\t\r \u00a0(2002).\t\r \u00a0309.\t\r \u00a0 A.\t\r \u00a0Frank,\t\r \u00a0P.\t\r \u00a0Pevzner,\t\r \u00a0PepNovo:\t\r \u00a0de\t\r \u00a0novo\t\r \u00a0peptide\t\r \u00a0sequencing\t\r \u00a0via\t\r \u00a0probabilistic\t\r \u00a0network\t\r \u00a0modeling.\t\r \u00a0Anal\t\r \u00a0Chem\t\r \u00a077,\t\r \u00a0964-\u00ad\u2010973\t\r \u00a0(2005).\t\r \u00a0310.\t\r \u00a0 A.\t\r \u00a0I.\t\r \u00a0Nesvizhskii,\t\r \u00a0F.\t\r \u00a0F.\t\r \u00a0Roos,\t\r \u00a0J.\t\r \u00a0Grossmann,\t\r \u00a0M.\t\r \u00a0Vogelzang,\t\r \u00a0J.\t\r \u00a0S.\t\r \u00a0Eddes,\t\r \u00a0W.\t\r \u00a0Gruissem,\t\r \u00a0S.\t\r \u00a0Baginsky,\t\r \u00a0R.\t\r \u00a0Aebersold,\t\r \u00a0Dynamic\t\r \u00a0spectrum\t\r \u00a0quality\t\r \u00a0assessment\t\r \u00a0and\t\r \u00a0iterative\t\r \u00a0computational\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0shotgun\t\r \u00a0proteomic\t\r \u00a0data:\t\r \u00a0toward\t\r \u00a0more\t\r \u00a0efficient\t\r \u00a0identification\t\r \u00a0of\t\r \u00a0post-\u00ad\u2010translational\t\r \u00a0modifications,\t\r \u00a0sequence\t\r \u00a0polymorphisms,\t\r \u00a0and\t\r \u00a0novel\t\r \u00a0peptides.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Proteomics\t\r \u00a05,\t\r \u00a0652-\u00ad\u2010670\t\r \u00a0(2006).\t\r \u00a0311.\t\r \u00a0 H.\t\r \u00a0Lam,\t\r \u00a0E.\t\r \u00a0W.\t\r \u00a0Deutsch,\t\r \u00a0J.\t\r \u00a0S.\t\r \u00a0Eddes,\t\r \u00a0J.\t\r \u00a0K.\t\r \u00a0Eng,\t\r \u00a0N.\t\r \u00a0King,\t\r \u00a0S.\t\r \u00a0E.\t\r \u00a0Stein,\t\r \u00a0R.\t\r \u00a0Aebersold,\t\r \u00a0Development\t\r \u00a0and\t\r \u00a0validation\t\r \u00a0of\t\r \u00a0a\t\r \u00a0spectral\t\r \u00a0library\t\r \u00a0searching\t\r \u00a0method\t\r \u00a0for\t\r \u00a0peptide\t\r \u00a0identification\t\r \u00a0from\t\r \u00a0MS\/MS.\t\r \u00a0Proteomics\t\r \u00a07,\t\r \u00a0655-\u00ad\u2010667\t\r \u00a0(2007).\t\r \u00a0312.\t\r \u00a0 X.\t\r \u00a0Wu,\t\r \u00a0C.\t\r \u00a0W.\t\r 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\u00a0Quantitative\t\r \u00a0mass\t\r \u00a0spectrometry\t\r \u00a0in\t\r \u00a0proteomics:\t\r \u00a0a\t\r \u00a0critical\t\r \u00a0review.\t\r \u00a0Anal\t\r \u00a0Bioanal\t\r \u00a0Chem\t\r \u00a0389,\t\r \u00a01017-\u00ad\u20101031\t\r \u00a0(2007).\t\r \u00a0319.\t\r \u00a0 S.\t\r \u00a0E.\t\r \u00a0Ong,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Mass\t\r \u00a0spectrometry-\u00ad\u2010based\t\r \u00a0proteomics\t\r \u00a0turns\t\r \u00a0quantitative.\t\r \u00a0 Nat\t\r \u00a0Chem\t\r \u00a0Biol\t\r \u00a01,\t\r \u00a0252-\u00ad\u2010262\t\r \u00a0(2005).\t\r \u00a0320.\t\r \u00a0 H.\t\r \u00a0Liu,\t\r \u00a0R.\t\r \u00a0G.\t\r \u00a0Sadygov,\t\r \u00a0J.\t\r \u00a0R.\t\r \u00a0Yates,\t\r \u00a03rd,\t\r \u00a0A\t\r \u00a0model\t\r \u00a0for\t\r \u00a0random\t\r \u00a0sampling\t\r \u00a0and\t\r \u00a0estimation\t\r \u00a0of\t\r \u00a0relative\t\r \u00a0protein\t\r \u00a0abundance\t\r \u00a0in\t\r \u00a0shotgun\t\r \u00a0proteomics.\t\r \u00a0Anal\t\r \u00a0Chem\t\r \u00a0 76,\t\r \u00a04193-\u00ad\u20104201\t\r \u00a0(2004).\t\r \u00a0321.\t\r \u00a0 Y.\t\r \u00a0Ishihama,\t\r \u00a0Y.\t\r \u00a0Oda,\t\r \u00a0T.\t\r \u00a0Tabata,\t\r \u00a0T.\t\r \u00a0Sato,\t\r \u00a0T.\t\r \u00a0Nagasu,\t\r \u00a0J.\t\r \u00a0Rappsilber,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Exponentially\t\r \u00a0modified\t\r \u00a0protein\t\r \u00a0abundance\t\r \u00a0index\t\r \u00a0(emPAI)\t\r \u00a0for\t\r \u00a0estimation\t\r \u00a0of\t\r \u00a0absolute\t\r \u00a0protein\t\r \u00a0amount\t\r \u00a0in\t\r \u00a0proteomics\t\r \u00a0by\t\r \u00a0the\t\r \u00a0number\t\r \u00a0of\t\r \u00a0sequenced\t\r \u00a0peptides\t\r \u00a0per\t\r \u00a0protein.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Proteomics\t\r \u00a04,\t\r \u00a01265-\u00ad\u20101272\t\r \u00a0(2005).\t\r \u00a0322.\t\r \u00a0 R.\t\r \u00a0D.\t\r \u00a0Smith,\t\r \u00a0G.\t\r \u00a0A.\t\r \u00a0Anderson,\t\r \u00a0M.\t\r \u00a0S.\t\r \u00a0Lipton,\t\r \u00a0L.\t\r \u00a0Pasa-\u00ad\u2010Tolic,\t\r \u00a0Y.\t\r \u00a0Shen,\t\r \u00a0T.\t\r \u00a0P.\t\r \u00a0Conrads,\t\r \u00a0T.\t\r \u00a0D.\t\r \u00a0Veenstra,\t\r \u00a0H.\t\r \u00a0R.\t\r \u00a0Udseth,\t\r \u00a0An\t\r \u00a0accurate\t\r \u00a0mass\t\r \u00a0tag\t\r \u00a0strategy\t\r \u00a0for\t\r \u00a0quantitative\t\r \u00a0and\t\r \u00a0high-\u00ad\u2010throughput\t\r \u00a0proteome\t\r \u00a0measurements.\t\r \u00a0Proteomics\t\r \u00a02,\t\r \u00a0513-\u00ad\u2010523\t\r \u00a0(2002).\t\r \u00a0323.\t\r \u00a0 M.\t\r \u00a0Ono,\t\r \u00a0M.\t\r \u00a0Shitashige,\t\r \u00a0K.\t\r \u00a0Honda,\t\r \u00a0T.\t\r \u00a0Isobe,\t\r \u00a0H.\t\r \u00a0Kuwabara,\t\r \u00a0H.\t\r \u00a0Matsuzuki,\t\r \u00a0S.\t\r \u00a0Hirohashi,\t\r \u00a0T.\t\r \u00a0Yamada,\t\r \u00a0Label-\u00ad\u2010free\t\r \u00a0quantitative\t\r \u00a0proteomics\t\r \u00a0using\t\r \u00a0large\t\r \u00a0peptide\t\r \u00a0data\t\r \u00a0sets\t\r \u00a0generated\t\r \u00a0by\t\r \u00a0nanoflow\t\r \u00a0liquid\t\r \u00a0chromatography\t\r \u00a0and\t\r \u00a0mass\t\r \u00a0spectrometry.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Proteomics\t\r \u00a05,\t\r \u00a01338-\u00ad\u20101347\t\r \u00a0(2006).\t\r \u00a0324.\t\r \u00a0 L.\t\r \u00a0Pa\u0161a-\u00ad\u2010Toli\u0107,\t\r \u00a0P.\t\r \u00a0K.\t\r \u00a0Jensen,\t\r \u00a0G.\t\r \u00a0A.\t\r \u00a0Anderson,\t\r \u00a0M.\t\r \u00a0S.\t\r \u00a0Lipton,\t\r \u00a0K.\t\r \u00a0K.\t\r \u00a0Peden,\t\r \u00a0S.\t\r \u00a0Martinovi\u0107,\t\r \u00a0N.\t\r \u00a0Toli\u0107,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Bruce,\t\r \u00a0R.\t\r \u00a0D.\t\r \u00a0Smith,\t\r \u00a0High\t\r \u00a0Throughput\t\r \u00a0Proteome-\u00ad\u2010Wide\t\r \u00a0Precision\t\r \u00a0Measurements\t\r \u00a0of\t\r \u00a0Protein\t\r \u00a0Expression\t\r \u00a0Using\t\r \u00a0Mass\t\r \u00a0Spectrometry.\t\r \u00a0 Journal\t\r \u00a0of\t\r \u00a0the\t\r \u00a0American\t\r \u00a0Chemical\t\r \u00a0Society\t\r \u00a0121,\t\r \u00a07949-\u00ad\u20107950\t\r \u00a0(1999).\t\r \u00a0325.\t\r \u00a0 J.\t\r \u00a0Krijgsveld,\t\r \u00a0R.\t\r \u00a0F.\t\r \u00a0Ketting,\t\r \u00a0T.\t\r \u00a0Mahmoudi,\t\r \u00a0J.\t\r \u00a0Johansen,\t\r \u00a0M.\t\r \u00a0Artal-\u00ad\u2010Sanz,\t\r \u00a0C.\t\r \u00a0P.\t\r \u00a0Verrijzer,\t\r \u00a0R.\t\r \u00a0H.\t\r \u00a0Plasterk,\t\r \u00a0A.\t\r \u00a0J.\t\r \u00a0Heck,\t\r \u00a0Metabolic\t\r \u00a0labeling\t\r \u00a0of\t\r \u00a0C.\t\r \u00a0elegans\t\r \u00a0and\t\r \u00a0D.\t\r \u00a0melanogaster\t\r \u00a0for\t\r \u00a0quantitative\t\r \u00a0proteomics.\t\r \u00a0Nat\t\r \u00a0Biotechnol\t\r \u00a021,\t\r \u00a0927-\u00ad\u2010931\t\r \u00a0(2003).\t\r \u00a0326.\t\r \u00a0 A.\t\r \u00a0Gruhler,\t\r \u00a0W.\t\r \u00a0X.\t\r \u00a0Schulze,\t\r \u00a0R.\t\r \u00a0Matthiesen,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0O.\t\r \u00a0N.\t\r \u00a0Jensen,\t\r \u00a0Stable\t\r \u00a0isotope\t\r \u00a0labeling\t\r \u00a0of\t\r \u00a0Arabidopsis\t\r \u00a0thaliana\t\r \u00a0cells\t\r \u00a0and\t\r \u00a0quantitative\t\r \u00a0proteomics\t\r \u00a0by\t\r \u00a0mass\t\r \u00a0spectrometry.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Proteomics\t\r \u00a04,\t\r \u00a01697-\u00ad\u20101709\t\r \u00a0(2005).\t\r \u00a0327.\t\r \u00a0 J.\t\r \u00a0W.\t\r \u00a0Gouw,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Tops,\t\r \u00a0P.\t\r \u00a0Mortensen,\t\r \u00a0A.\t\r \u00a0J.\t\r \u00a0Heck,\t\r \u00a0J.\t\r \u00a0Krijgsveld,\t\r \u00a0Optimizing\t\r \u00a0identification\t\r \u00a0and\t\r \u00a0quantitation\t\r \u00a0of\t\r \u00a015N-\u00ad\u2010labeled\t\r \u00a0proteins\t\r \u00a0in\t\r \u00a0comparative\t\r \u00a0proteomics.\t\r \u00a0Anal\t\r \u00a0Chem\t\r \u00a080,\t\r \u00a07796-\u00ad\u20107803\t\r \u00a0(2008).\t\r \u00a0328.\t\r \u00a0 J.\t\r \u00a0W.\t\r \u00a0Gouw,\t\r \u00a0M.\t\r \u00a0W.\t\r \u00a0Pinkse,\t\r \u00a0H.\t\r \u00a0R.\t\r \u00a0Vos,\t\r \u00a0Y.\t\r \u00a0Moshkin,\t\r \u00a0C.\t\r \u00a0P.\t\r \u00a0Verrijzer,\t\r \u00a0A.\t\r \u00a0J.\t\r \u00a0Heck,\t\r \u00a0J.\t\r \u00a0Krijgsveld,\t\r \u00a0In\t\r \u00a0vivo\t\r \u00a0stable\t\r \u00a0isotope\t\r \u00a0labeling\t\r \u00a0of\t\r \u00a0fruit\t\r \u00a0flies\t\r \u00a0reveals\t\r \u00a0post-\u00ad\u2010transcriptional\t\r \u00a0regulation\t\r \u00a0in\t\r \u00a0the\t\r \u00a0maternal-\u00ad\u2010to-\u00ad\u2010zygotic\t\r \u00a0transition.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0 Proteomics\t\r \u00a08,\t\r \u00a01566-\u00ad\u20101578\t\r \u00a0(2009).\t\r \u00a0329.\t\r \u00a0 S.\t\r \u00a0E.\t\r \u00a0Ong,\t\r \u00a0B.\t\r \u00a0Blagoev,\t\r \u00a0I.\t\r \u00a0Kratchmarova,\t\r \u00a0D.\t\r \u00a0B.\t\r \u00a0Kristensen,\t\r \u00a0H.\t\r \u00a0Steen,\t\r \u00a0A.\t\r \u00a0Pandey,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Stable\t\r \u00a0isotope\t\r \u00a0labeling\t\r \u00a0by\t\r \u00a0amino\t\r \u00a0acids\t\r \u00a0in\t\r \u00a0cell\t\r \u00a0culture,\t\r \u00a0SILAC,\t\r \u00a0as\t\r \u00a0a\t\r \u00a0 \t\r \u00a0\t\r \u00a0 168\t\r \u00a0 simple\t\r \u00a0and\t\r \u00a0accurate\t\r \u00a0approach\t\r \u00a0to\t\r \u00a0expression\t\r \u00a0proteomics.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Proteomics\t\r \u00a0 1,\t\r \u00a0376-\u00ad\u2010386\t\r \u00a0(2002).\t\r \u00a0330.\t\r \u00a0 S.\t\r \u00a0E.\t\r \u00a0Ong,\t\r \u00a0I.\t\r \u00a0Kratchmarova,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Properties\t\r \u00a0of\t\r \u00a013C-\u00ad\u2010substituted\t\r \u00a0arginine\t\r \u00a0in\t\r \u00a0stable\t\r \u00a0isotope\t\r \u00a0labeling\t\r \u00a0by\t\r \u00a0amino\t\r \u00a0acids\t\r \u00a0in\t\r \u00a0cell\t\r \u00a0culture\t\r \u00a0(SILAC).\t\r \u00a0J\t\r \u00a0Proteome\t\r \u00a0Res\t\r \u00a0 2,\t\r \u00a0173-\u00ad\u2010181\t\r \u00a0(2003).\t\r \u00a0331.\t\r \u00a0 B.\t\r \u00a0Blagoev,\t\r \u00a0S.\t\r \u00a0E.\t\r \u00a0Ong,\t\r \u00a0I.\t\r \u00a0Kratchmarova,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Temporal\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0phosphotyrosine-\u00ad\u2010dependent\t\r \u00a0signaling\t\r \u00a0networks\t\r \u00a0by\t\r \u00a0quantitative\t\r \u00a0proteomics.\t\r \u00a0 Nat\t\r \u00a0Biotechnol\t\r \u00a022,\t\r \u00a01139-\u00ad\u20101145\t\r \u00a0(2004).\t\r \u00a0332.\t\r \u00a0 L.\t\r \u00a0M.\t\r \u00a0de\t\r \u00a0Godoy,\t\r \u00a0J.\t\r \u00a0V.\t\r \u00a0Olsen,\t\r \u00a0J.\t\r \u00a0Cox,\t\r \u00a0M.\t\r \u00a0L.\t\r \u00a0Nielsen,\t\r \u00a0N.\t\r \u00a0C.\t\r \u00a0Hubner,\t\r \u00a0F.\t\r \u00a0Frohlich,\t\r \u00a0T.\t\r \u00a0C.\t\r \u00a0Walther,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Comprehensive\t\r \u00a0mass-\u00ad\u2010spectrometry-\u00ad\u2010based\t\r \u00a0proteome\t\r \u00a0quantification\t\r \u00a0of\t\r \u00a0haploid\t\r \u00a0versus\t\r \u00a0diploid\t\r \u00a0yeast.\t\r \u00a0Nature\t\r \u00a0455,\t\r \u00a01251-\u00ad\u20101254\t\r \u00a0(2008).\t\r \u00a0333.\t\r \u00a0 C.\t\r \u00a0L.\t\r \u00a0de\t\r \u00a0Hoog,\t\r \u00a0L.\t\r \u00a0J.\t\r \u00a0Foster,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0RNA\t\r \u00a0and\t\r \u00a0RNA\t\r \u00a0binding\t\r \u00a0proteins\t\r \u00a0participate\t\r \u00a0in\t\r \u00a0early\t\r \u00a0stages\t\r \u00a0of\t\r \u00a0cell\t\r \u00a0spreading\t\r \u00a0through\t\r \u00a0spreading\t\r \u00a0initiation\t\r \u00a0centers.\t\r \u00a0Cell\t\r \u00a0 117,\t\r \u00a0649-\u00ad\u2010662\t\r \u00a0(2004).\t\r \u00a0334.\t\r \u00a0 J.\t\r \u00a0V.\t\r \u00a0Olsen,\t\r \u00a0M.\t\r \u00a0Vermeulen,\t\r \u00a0A.\t\r \u00a0Santamaria,\t\r \u00a0C.\t\r \u00a0Kumar,\t\r \u00a0M.\t\r \u00a0L.\t\r \u00a0Miller,\t\r \u00a0L.\t\r \u00a0J.\t\r \u00a0Jensen,\t\r \u00a0F.\t\r \u00a0Gnad,\t\r \u00a0J.\t\r \u00a0Cox,\t\r \u00a0T.\t\r \u00a0S.\t\r \u00a0Jensen,\t\r \u00a0E.\t\r \u00a0A.\t\r \u00a0Nigg,\t\r \u00a0S.\t\r \u00a0Brunak,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Quantitative\t\r \u00a0phosphoproteomics\t\r \u00a0reveals\t\r \u00a0widespread\t\r \u00a0full\t\r \u00a0phosphorylation\t\r \u00a0site\t\r \u00a0occupancy\t\r \u00a0during\t\r \u00a0mitosis.\t\r \u00a0Sci\t\r \u00a0Signal\t\r \u00a03,\t\r \u00a0ra3\t\r \u00a0(2010).\t\r \u00a0335.\t\r \u00a0 M.\t\r \u00a0P.\t\r \u00a0Stokes,\t\r \u00a0J.\t\r \u00a0Rush,\t\r \u00a0J.\t\r \u00a0Macneill,\t\r \u00a0J.\t\r \u00a0M.\t\r \u00a0Ren,\t\r \u00a0K.\t\r \u00a0Sprott,\t\r \u00a0J.\t\r \u00a0Nardone,\t\r \u00a0V.\t\r \u00a0Yang,\t\r \u00a0S.\t\r \u00a0A.\t\r \u00a0Beausoleil,\t\r \u00a0S.\t\r \u00a0P.\t\r \u00a0Gygi,\t\r \u00a0M.\t\r \u00a0Livingstone,\t\r \u00a0H.\t\r \u00a0Zhang,\t\r \u00a0R.\t\r \u00a0D.\t\r \u00a0Polakiewicz,\t\r \u00a0M.\t\r \u00a0J.\t\r \u00a0Comb,\t\r \u00a0Profiling\t\r \u00a0of\t\r \u00a0UV-\u00ad\u2010induced\t\r \u00a0ATM\/ATR\t\r \u00a0signaling\t\r \u00a0pathways.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0 A\t\r \u00a0104,\t\r \u00a019855-\u00ad\u201019860\t\r \u00a0(2007).\t\r \u00a0336.\t\r \u00a0 L.\t\r \u00a0J.\t\r \u00a0Foster,\t\r \u00a0A.\t\r \u00a0Rudich,\t\r \u00a0I.\t\r \u00a0Talior,\t\r \u00a0N.\t\r \u00a0Patel,\t\r \u00a0X.\t\r \u00a0Huang,\t\r \u00a0L.\t\r \u00a0M.\t\r \u00a0Furtado,\t\r \u00a0P.\t\r \u00a0J.\t\r \u00a0Bilan,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0A.\t\r \u00a0Klip,\t\r \u00a0Insulin-\u00ad\u2010dependent\t\r \u00a0interactions\t\r \u00a0of\t\r \u00a0proteins\t\r \u00a0with\t\r \u00a0GLUT4\t\r \u00a0revealed\t\r \u00a0through\t\r \u00a0stable\t\r \u00a0isotope\t\r \u00a0labeling\t\r \u00a0by\t\r \u00a0amino\t\r \u00a0acids\t\r \u00a0in\t\r \u00a0cell\t\r \u00a0culture\t\r \u00a0(SILAC).\t\r \u00a0J\t\r \u00a0Proteome\t\r \u00a0Res\t\r \u00a05,\t\r \u00a064-\u00ad\u201075\t\r \u00a0(2006).\t\r \u00a0337.\t\r \u00a0 B.\t\r \u00a0Blagoev,\t\r \u00a0I.\t\r \u00a0Kratchmarova,\t\r \u00a0S.\t\r \u00a0E.\t\r \u00a0Ong,\t\r \u00a0M.\t\r \u00a0Nielsen,\t\r \u00a0L.\t\r \u00a0J.\t\r \u00a0Foster,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0A\t\r \u00a0proteomics\t\r \u00a0strategy\t\r \u00a0to\t\r \u00a0elucidate\t\r \u00a0functional\t\r \u00a0protein-\u00ad\u2010protein\t\r \u00a0interactions\t\r \u00a0applied\t\r \u00a0to\t\r \u00a0EGF\t\r \u00a0signaling.\t\r \u00a0Nat\t\r \u00a0Biotechnol\t\r \u00a021,\t\r \u00a0315-\u00ad\u2010318\t\r \u00a0(2003).\t\r \u00a0338.\t\r \u00a0 M.\t\r \u00a0Kruger,\t\r \u00a0M.\t\r \u00a0Moser,\t\r \u00a0S.\t\r \u00a0Ussar,\t\r \u00a0I.\t\r \u00a0Thievessen,\t\r \u00a0C.\t\r \u00a0A.\t\r \u00a0Luber,\t\r \u00a0F.\t\r \u00a0Forner,\t\r \u00a0S.\t\r \u00a0Schmidt,\t\r \u00a0S.\t\r \u00a0Zanivan,\t\r \u00a0R.\t\r \u00a0Fassler,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0SILAC\t\r \u00a0mouse\t\r \u00a0for\t\r \u00a0quantitative\t\r \u00a0proteomics\t\r \u00a0uncovers\t\r \u00a0kindlin-\u00ad\u20103\t\r \u00a0as\t\r \u00a0an\t\r \u00a0essential\t\r \u00a0factor\t\r \u00a0for\t\r \u00a0red\t\r \u00a0blood\t\r \u00a0cell\t\r \u00a0function.\t\r \u00a0Cell\t\r \u00a0134,\t\r \u00a0353-\u00ad\u2010364\t\r \u00a0(2008).\t\r \u00a0339.\t\r \u00a0 S.\t\r \u00a0P.\t\r \u00a0Gygi,\t\r \u00a0B.\t\r \u00a0Rist,\t\r \u00a0S.\t\r \u00a0A.\t\r \u00a0Gerber,\t\r \u00a0F.\t\r \u00a0Turecek,\t\r \u00a0M.\t\r \u00a0H.\t\r \u00a0Gelb,\t\r \u00a0R.\t\r \u00a0Aebersold,\t\r \u00a0Quantitative\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0complex\t\r \u00a0protein\t\r \u00a0mixtures\t\r \u00a0using\t\r \u00a0isotope-\u00ad\u2010coded\t\r \u00a0affinity\t\r \u00a0tags.\t\r \u00a0Nat\t\r \u00a0Biotechnol\t\r \u00a017,\t\r \u00a0994-\u00ad\u2010999\t\r \u00a0(1999).\t\r \u00a0340.\t\r \u00a0 K.\t\r \u00a0C.\t\r \u00a0Hansen,\t\r \u00a0G.\t\r \u00a0Schmitt-\u00ad\u2010Ulms,\t\r \u00a0R.\t\r \u00a0J.\t\r \u00a0Chalkley,\t\r \u00a0J.\t\r \u00a0Hirsch,\t\r \u00a0M.\t\r \u00a0A.\t\r \u00a0Baldwin,\t\r \u00a0A.\t\r \u00a0L.\t\r \u00a0Burlingame,\t\r \u00a0Mass\t\r \u00a0spectrometric\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0protein\t\r \u00a0mixtures\t\r \u00a0at\t\r \u00a0low\t\r \u00a0levels\t\r \u00a0using\t\r \u00a0cleavable\t\r \u00a013C-\u00ad\u2010isotope-\u00ad\u2010coded\t\r \u00a0affinity\t\r \u00a0tag\t\r \u00a0and\t\r \u00a0multidimensional\t\r \u00a0chromatography.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Proteomics\t\r \u00a02,\t\r \u00a0299-\u00ad\u2010314\t\r \u00a0(2003).\t\r \u00a0341.\t\r \u00a0 J.\t\r \u00a0Li,\t\r \u00a0H.\t\r \u00a0Steen,\t\r \u00a0S.\t\r \u00a0P.\t\r \u00a0Gygi,\t\r \u00a0Protein\t\r \u00a0profiling\t\r \u00a0with\t\r \u00a0cleavable\t\r \u00a0isotope-\u00ad\u2010coded\t\r \u00a0affinity\t\r \u00a0tag\t\r \u00a0(cICAT)\t\r \u00a0reagents:\t\r \u00a0the\t\r \u00a0yeast\t\r \u00a0salinity\t\r \u00a0stress\t\r \u00a0response.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0 Proteomics\t\r \u00a02,\t\r \u00a01198-\u00ad\u20101204\t\r \u00a0(2003).\t\r \u00a0342.\t\r \u00a0 J.\t\r \u00a0L.\t\r \u00a0Hsu,\t\r \u00a0S.\t\r \u00a0Y.\t\r \u00a0Huang,\t\r \u00a0N.\t\r \u00a0H.\t\r \u00a0Chow,\t\r \u00a0S.\t\r \u00a0H.\t\r \u00a0Chen,\t\r \u00a0Stable-\u00ad\u2010isotope\t\r \u00a0dimethyl\t\r \u00a0labeling\t\r \u00a0for\t\r \u00a0quantitative\t\r \u00a0proteomics.\t\r \u00a0Anal\t\r \u00a0Chem\t\r \u00a075,\t\r \u00a06843-\u00ad\u20106852\t\r \u00a0(2003).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 169\t\r \u00a0 343.\t\r \u00a0 P.\t\r \u00a0J.\t\r \u00a0Boersema,\t\r \u00a0T.\t\r \u00a0T.\t\r \u00a0Aye,\t\r \u00a0T.\t\r \u00a0A.\t\r \u00a0van\t\r \u00a0Veen,\t\r \u00a0A.\t\r \u00a0J.\t\r \u00a0Heck,\t\r \u00a0S.\t\r \u00a0Mohammed,\t\r \u00a0Triplex\t\r \u00a0protein\t\r \u00a0quantification\t\r \u00a0based\t\r \u00a0on\t\r \u00a0stable\t\r \u00a0isotope\t\r \u00a0labeling\t\r \u00a0by\t\r \u00a0peptide\t\r \u00a0dimethylation\t\r \u00a0applied\t\r \u00a0to\t\r \u00a0cell\t\r \u00a0and\t\r \u00a0tissue\t\r \u00a0lysates.\t\r \u00a0Proteomics\t\r \u00a08,\t\r \u00a04624-\u00ad\u20104632\t\r \u00a0(2008).\t\r \u00a0344.\t\r \u00a0 P.\t\r \u00a0L.\t\r \u00a0Ross,\t\r \u00a0Y.\t\r \u00a0N.\t\r \u00a0Huang,\t\r \u00a0J.\t\r \u00a0N.\t\r \u00a0Marchese,\t\r \u00a0B.\t\r \u00a0Williamson,\t\r \u00a0K.\t\r \u00a0Parker,\t\r \u00a0S.\t\r \u00a0Hattan,\t\r \u00a0N.\t\r \u00a0Khainovski,\t\r \u00a0S.\t\r \u00a0Pillai,\t\r \u00a0S.\t\r \u00a0Dey,\t\r \u00a0S.\t\r \u00a0Daniels,\t\r \u00a0S.\t\r \u00a0Purkayastha,\t\r \u00a0P.\t\r \u00a0Juhasz,\t\r \u00a0S.\t\r \u00a0Martin,\t\r \u00a0M.\t\r \u00a0Bartlet-\u00ad\u2010Jones,\t\r \u00a0F.\t\r \u00a0He,\t\r \u00a0A.\t\r \u00a0Jacobson,\t\r \u00a0D.\t\r \u00a0J.\t\r \u00a0Pappin,\t\r \u00a0Multiplexed\t\r \u00a0protein\t\r \u00a0quantitation\t\r \u00a0in\t\r \u00a0Saccharomyces\t\r \u00a0cerevisiae\t\r \u00a0using\t\r \u00a0amine-\u00ad\u2010reactive\t\r \u00a0isobaric\t\r \u00a0tagging\t\r \u00a0reagents.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Proteomics\t\r \u00a03,\t\r \u00a01154-\u00ad\u20101169\t\r \u00a0(2004).\t\r \u00a0345.\t\r \u00a0 L.\t\r \u00a0Choe,\t\r \u00a0M.\t\r \u00a0D'Ascenzo,\t\r \u00a0N.\t\r \u00a0R.\t\r \u00a0Relkin,\t\r \u00a0D.\t\r \u00a0Pappin,\t\r \u00a0P.\t\r \u00a0Ross,\t\r \u00a0B.\t\r \u00a0Williamson,\t\r \u00a0S.\t\r \u00a0Guertin,\t\r \u00a0P.\t\r \u00a0Pribil,\t\r \u00a0K.\t\r \u00a0H.\t\r \u00a0Lee,\t\r \u00a08-\u00ad\u2010plex\t\r \u00a0quantitation\t\r \u00a0of\t\r \u00a0changes\t\r \u00a0in\t\r \u00a0cerebrospinal\t\r \u00a0fluid\t\r \u00a0protein\t\r \u00a0expression\t\r \u00a0in\t\r \u00a0subjects\t\r \u00a0undergoing\t\r \u00a0intravenous\t\r \u00a0immunoglobulin\t\r \u00a0treatment\t\r \u00a0for\t\r \u00a0Alzheimer's\t\r \u00a0disease.\t\r \u00a0Proteomics\t\r \u00a07,\t\r \u00a03651-\u00ad\u20103660\t\r \u00a0(2007).\t\r \u00a0346.\t\r \u00a0 S.\t\r \u00a0A.\t\r \u00a0Gerber,\t\r \u00a0J.\t\r \u00a0Rush,\t\r \u00a0O.\t\r \u00a0Stemman,\t\r \u00a0M.\t\r \u00a0W.\t\r \u00a0Kirschner,\t\r \u00a0S.\t\r \u00a0P.\t\r \u00a0Gygi,\t\r \u00a0Absolute\t\r \u00a0quantification\t\r \u00a0of\t\r \u00a0proteins\t\r \u00a0and\t\r \u00a0phosphoproteins\t\r \u00a0from\t\r \u00a0cell\t\r \u00a0lysates\t\r \u00a0by\t\r \u00a0tandem\t\r \u00a0MS.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a0100,\t\r \u00a06940-\u00ad\u20106945\t\r \u00a0(2003).\t\r \u00a0347.\t\r \u00a0 D.\t\r \u00a0S.\t\r \u00a0Kirkpatrick,\t\r \u00a0S.\t\r \u00a0A.\t\r \u00a0Gerber,\t\r \u00a0S.\t\r \u00a0P.\t\r \u00a0Gygi,\t\r \u00a0The\t\r \u00a0absolute\t\r \u00a0quantification\t\r \u00a0strategy:\t\r \u00a0a\t\r \u00a0general\t\r \u00a0procedure\t\r \u00a0for\t\r \u00a0the\t\r \u00a0quantification\t\r \u00a0of\t\r \u00a0proteins\t\r \u00a0and\t\r \u00a0post-\u00ad\u2010translational\t\r \u00a0modifications.\t\r \u00a0Methods\t\r \u00a035,\t\r \u00a0265-\u00ad\u2010273\t\r \u00a0(2005).\t\r \u00a0348.\t\r \u00a0 A.\t\r \u00a0Wolf-\u00ad\u2010Yadlin,\t\r \u00a0S.\t\r \u00a0Hautaniemi,\t\r \u00a0D.\t\r \u00a0A.\t\r \u00a0Lauffenburger,\t\r \u00a0F.\t\r \u00a0M.\t\r \u00a0White,\t\r \u00a0Multiple\t\r \u00a0reaction\t\r \u00a0monitoring\t\r \u00a0for\t\r \u00a0robust\t\r \u00a0quantitative\t\r \u00a0proteomic\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0cellular\t\r \u00a0signaling\t\r \u00a0networks.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a0104,\t\r \u00a05860-\u00ad\u20105865\t\r \u00a0(2007).\t\r \u00a0349.\t\r \u00a0 R.\t\r \u00a0J.\t\r \u00a0Beynon,\t\r \u00a0M.\t\r \u00a0K.\t\r \u00a0Doherty,\t\r \u00a0J.\t\r \u00a0M.\t\r \u00a0Pratt,\t\r \u00a0S.\t\r \u00a0J.\t\r \u00a0Gaskell,\t\r \u00a0Multiplexed\t\r \u00a0absolute\t\r \u00a0quantification\t\r \u00a0in\t\r \u00a0proteomics\t\r \u00a0using\t\r \u00a0artificial\t\r \u00a0QCAT\t\r \u00a0proteins\t\r \u00a0of\t\r \u00a0concatenated\t\r \u00a0signature\t\r \u00a0peptides.\t\r \u00a0Nat\t\r \u00a0Methods\t\r \u00a02,\t\r \u00a0587-\u00ad\u2010589\t\r \u00a0(2005).\t\r \u00a0350.\t\r \u00a0 P.\t\r \u00a0Mortensen,\t\r \u00a0J.\t\r \u00a0W.\t\r \u00a0Gouw,\t\r \u00a0J.\t\r \u00a0V.\t\r \u00a0Olsen,\t\r \u00a0S.\t\r \u00a0E.\t\r \u00a0Ong,\t\r \u00a0K.\t\r \u00a0T.\t\r \u00a0Rigbolt,\t\r \u00a0J.\t\r \u00a0Bunkenborg,\t\r \u00a0J.\t\r \u00a0Cox,\t\r \u00a0L.\t\r \u00a0J.\t\r \u00a0Foster,\t\r \u00a0A.\t\r \u00a0J.\t\r \u00a0Heck,\t\r \u00a0B.\t\r \u00a0Blagoev,\t\r \u00a0J.\t\r \u00a0S.\t\r \u00a0Andersen,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0MSQuant,\t\r \u00a0an\t\r \u00a0open\t\r \u00a0source\t\r \u00a0platform\t\r \u00a0for\t\r \u00a0mass\t\r \u00a0spectrometry-\u00ad\u2010based\t\r \u00a0quantitative\t\r \u00a0proteomics.\t\r \u00a0 J\t\r \u00a0Proteome\t\r \u00a0Res\t\r \u00a09,\t\r \u00a0393-\u00ad\u2010403\t\r \u00a0(2010).\t\r \u00a0351.\t\r \u00a0 J.\t\r \u00a0Cox,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0MaxQuant\t\r \u00a0enables\t\r \u00a0high\t\r \u00a0peptide\t\r \u00a0identification\t\r \u00a0rates,\t\r \u00a0individualized\t\r \u00a0p.p.b.-\u00ad\u2010range\t\r \u00a0mass\t\r \u00a0accuracies\t\r \u00a0and\t\r \u00a0proteome-\u00ad\u2010wide\t\r \u00a0protein\t\r \u00a0quantification.\t\r \u00a0Nat\t\r \u00a0Biotechnol\t\r \u00a026,\t\r \u00a01367-\u00ad\u20101372\t\r \u00a0(2008).\t\r \u00a0352.\t\r \u00a0 M.\t\r \u00a0Clamp,\t\r \u00a0B.\t\r \u00a0Fry,\t\r \u00a0M.\t\r \u00a0Kamal,\t\r \u00a0X.\t\r \u00a0Xie,\t\r \u00a0J.\t\r \u00a0Cuff,\t\r \u00a0M.\t\r \u00a0F.\t\r \u00a0Lin,\t\r \u00a0M.\t\r \u00a0Kellis,\t\r \u00a0K.\t\r \u00a0Lindblad-\u00ad\u2010Toh,\t\r \u00a0E.\t\r \u00a0S.\t\r \u00a0Lander,\t\r \u00a0Distinguishing\t\r \u00a0protein-\u00ad\u2010coding\t\r \u00a0and\t\r \u00a0noncoding\t\r \u00a0genes\t\r \u00a0in\t\r \u00a0the\t\r \u00a0human\t\r \u00a0genome.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a0104,\t\r \u00a019428-\u00ad\u201019433\t\r \u00a0(2007).\t\r \u00a0353.\t\r \u00a0 J.\t\r \u00a0Cox,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Is\t\r \u00a0proteomics\t\r \u00a0the\t\r \u00a0new\t\r \u00a0genomics?\t\r \u00a0Cell\t\r \u00a0130,\t\r \u00a0395-\u00ad\u2010398\t\r \u00a0(2007).\t\r \u00a0354.\t\r \u00a0 G.\t\r \u00a0L.\t\r \u00a0Corthals,\t\r \u00a0V.\t\r \u00a0C.\t\r \u00a0Wasinger,\t\r \u00a0D.\t\r \u00a0F.\t\r \u00a0Hochstrasser,\t\r \u00a0J.\t\r \u00a0C.\t\r \u00a0Sanchez,\t\r \u00a0The\t\r \u00a0dynamic\t\r \u00a0range\t\r \u00a0of\t\r \u00a0protein\t\r \u00a0expression:\t\r \u00a0a\t\r \u00a0challenge\t\r \u00a0for\t\r \u00a0proteomic\t\r \u00a0research.\t\r \u00a0 Electrophoresis\t\r \u00a021,\t\r \u00a01104-\u00ad\u20101115\t\r \u00a0(2000).\t\r \u00a0355.\t\r \u00a0 J.\t\r \u00a0V.\t\r \u00a0Olsen,\t\r \u00a0J.\t\r \u00a0C.\t\r \u00a0Schwartz,\t\r \u00a0J.\t\r \u00a0Griep-\u00ad\u2010Raming,\t\r \u00a0M.\t\r \u00a0L.\t\r \u00a0Nielsen,\t\r \u00a0E.\t\r \u00a0Damoc,\t\r \u00a0E.\t\r \u00a0Denisov,\t\r \u00a0O.\t\r \u00a0Lange,\t\r \u00a0P.\t\r \u00a0Remes,\t\r \u00a0D.\t\r \u00a0Taylor,\t\r \u00a0M.\t\r \u00a0Splendore,\t\r \u00a0E.\t\r \u00a0R.\t\r \u00a0Wouters,\t\r \u00a0M.\t\r \u00a0Senko,\t\r \u00a0A.\t\r \u00a0Makarov,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0S.\t\r \u00a0Horning,\t\r \u00a0A\t\r \u00a0dual\t\r \u00a0pressure\t\r \u00a0linear\t\r \u00a0ion\t\r \u00a0trap\t\r \u00a0Orbitrap\t\r \u00a0instrument\t\r \u00a0with\t\r \u00a0very\t\r \u00a0high\t\r \u00a0sequencing\t\r \u00a0speed.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Proteomics\t\r \u00a08,\t\r \u00a02759-\u00ad\u20102769\t\r \u00a0(2009).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 170\t\r \u00a0 356.\t\r \u00a0 M.\t\r \u00a0P.\t\r \u00a0Washburn,\t\r \u00a0D.\t\r \u00a0Wolters,\t\r \u00a0J.\t\r \u00a0R.\t\r \u00a0Yates,\t\r \u00a03rd,\t\r \u00a0Large-\u00ad\u2010scale\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0the\t\r \u00a0yeast\t\r \u00a0proteome\t\r \u00a0by\t\r \u00a0multidimensional\t\r \u00a0protein\t\r \u00a0identification\t\r \u00a0technology.\t\r \u00a0Nat\t\r \u00a0 Biotechnol\t\r \u00a019,\t\r \u00a0242-\u00ad\u2010247\t\r \u00a0(2001).\t\r \u00a0357.\t\r \u00a0 J.\t\r \u00a0Peng,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Elias,\t\r \u00a0C.\t\r \u00a0C.\t\r \u00a0Thoreen,\t\r \u00a0L.\t\r \u00a0J.\t\r \u00a0Licklider,\t\r \u00a0S.\t\r \u00a0P.\t\r \u00a0Gygi,\t\r \u00a0Evaluation\t\r \u00a0of\t\r \u00a0multidimensional\t\r \u00a0chromatography\t\r \u00a0coupled\t\r \u00a0with\t\r \u00a0tandem\t\r \u00a0mass\t\r \u00a0spectrometry\t\r \u00a0(LC\/LC-\u00ad\u2010MS\/MS)\t\r \u00a0for\t\r \u00a0large-\u00ad\u2010scale\t\r \u00a0protein\t\r \u00a0analysis:\t\r \u00a0the\t\r \u00a0yeast\t\r \u00a0proteome.\t\r \u00a0J\t\r \u00a0 Proteome\t\r \u00a0Res\t\r \u00a02,\t\r \u00a043-\u00ad\u201050\t\r \u00a0(2003).\t\r \u00a0358.\t\r \u00a0 Y.\t\r \u00a0Ishihama,\t\r \u00a0J.\t\r \u00a0Rappsilber,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Modular\t\r \u00a0stop\t\r \u00a0and\t\r \u00a0go\t\r \u00a0extraction\t\r \u00a0tips\t\r \u00a0with\t\r \u00a0stacked\t\r \u00a0disks\t\r \u00a0for\t\r \u00a0parallel\t\r \u00a0and\t\r \u00a0multidimensional\t\r \u00a0Peptide\t\r \u00a0fractionation\t\r \u00a0in\t\r \u00a0proteomics.\t\r \u00a0J\t\r \u00a0Proteome\t\r \u00a0Res\t\r \u00a05,\t\r \u00a0988-\u00ad\u2010994\t\r \u00a0(2006).\t\r \u00a0359.\t\r \u00a0 G.\t\r \u00a0Han,\t\r \u00a0M.\t\r \u00a0Ye,\t\r \u00a0H.\t\r \u00a0Zhou,\t\r \u00a0X.\t\r \u00a0Jiang,\t\r \u00a0S.\t\r \u00a0Feng,\t\r \u00a0R.\t\r \u00a0Tian,\t\r \u00a0D.\t\r \u00a0Wan,\t\r \u00a0H.\t\r \u00a0Zou,\t\r \u00a0J.\t\r \u00a0Gu,\t\r \u00a0Large-\u00ad\u2010scale\t\r \u00a0phosphoproteome\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0human\t\r \u00a0liver\t\r \u00a0tissue\t\r \u00a0by\t\r \u00a0enrichment\t\r \u00a0and\t\r \u00a0fractionation\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0with\t\r \u00a0strong\t\r \u00a0anion\t\r \u00a0exchange\t\r \u00a0chromatography.\t\r \u00a0Proteomics\t\r \u00a08,\t\r \u00a01346-\u00ad\u20101361\t\r \u00a0(2008).\t\r \u00a0360.\t\r \u00a0 G.\t\r \u00a0Alvarez-\u00ad\u2010Manilla,\t\r \u00a0J.\t\r \u00a0Atwood,\t\r \u00a03rd,\t\r \u00a0Y.\t\r \u00a0Guo,\t\r \u00a0N.\t\r \u00a0L.\t\r \u00a0Warren,\t\r \u00a0R.\t\r \u00a0Orlando,\t\r \u00a0M.\t\r \u00a0Pierce,\t\r \u00a0Tools\t\r \u00a0for\t\r \u00a0glycoproteomic\t\r \u00a0analysis:\t\r \u00a0size\t\r \u00a0exclusion\t\r \u00a0chromatography\t\r \u00a0facilitates\t\r \u00a0identification\t\r \u00a0of\t\r \u00a0tryptic\t\r \u00a0glycopeptides\t\r \u00a0with\t\r \u00a0N-\u00ad\u2010linked\t\r \u00a0glycosylation\t\r \u00a0sites.\t\r \u00a0J\t\r \u00a0 Proteome\t\r \u00a0Res\t\r \u00a05,\t\r \u00a0701-\u00ad\u2010708\t\r \u00a0(2006).\t\r \u00a0361.\t\r \u00a0 A.\t\r \u00a0Thorsell,\t\r \u00a0E.\t\r \u00a0Portelius,\t\r \u00a0K.\t\r \u00a0Blennow,\t\r \u00a0A.\t\r \u00a0Westman-\u00ad\u2010Brinkmalm,\t\r \u00a0Evaluation\t\r \u00a0of\t\r \u00a0sample\t\r \u00a0fractionation\t\r \u00a0using\t\r \u00a0micro-\u00ad\u2010scale\t\r \u00a0liquid-\u00ad\u2010phase\t\r \u00a0isoelectric\t\r \u00a0focusing\t\r \u00a0on\t\r \u00a0mass\t\r \u00a0spectrometric\t\r \u00a0identification\t\r \u00a0and\t\r \u00a0quantitation\t\r \u00a0of\t\r \u00a0proteins\t\r \u00a0in\t\r \u00a0a\t\r \u00a0SILAC\t\r \u00a0experiment.\t\r \u00a0Rapid\t\r \u00a0Commun\t\r \u00a0Mass\t\r \u00a0Spectrom\t\r \u00a021,\t\r \u00a0771-\u00ad\u2010778\t\r \u00a0(2007).\t\r \u00a0362.\t\r \u00a0 N.\t\r \u00a0C.\t\r \u00a0Hubner,\t\r \u00a0S.\t\r \u00a0Ren,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Peptide\t\r \u00a0separation\t\r \u00a0with\t\r \u00a0immobilized\t\r \u00a0pI\t\r \u00a0strips\t\r \u00a0is\t\r \u00a0an\t\r \u00a0attractive\t\r \u00a0alternative\t\r \u00a0to\t\r \u00a0in-\u00ad\u2010gel\t\r \u00a0protein\t\r \u00a0digestion\t\r \u00a0for\t\r \u00a0proteome\t\r \u00a0analysis.\t\r \u00a0 Proteomics\t\r \u00a08,\t\r \u00a04862-\u00ad\u20104872\t\r \u00a0(2008).\t\r \u00a0363.\t\r \u00a0 E.\t\r \u00a0Lasonder,\t\r \u00a0Y.\t\r \u00a0Ishihama,\t\r \u00a0J.\t\r \u00a0S.\t\r \u00a0Andersen,\t\r \u00a0A.\t\r \u00a0M.\t\r \u00a0Vermunt,\t\r \u00a0A.\t\r \u00a0Pain,\t\r \u00a0R.\t\r \u00a0W.\t\r \u00a0Sauerwein,\t\r \u00a0W.\t\r \u00a0M.\t\r \u00a0Eling,\t\r \u00a0N.\t\r \u00a0Hall,\t\r \u00a0A.\t\r \u00a0P.\t\r \u00a0Waters,\t\r \u00a0H.\t\r \u00a0G.\t\r \u00a0Stunnenberg,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Analysis\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Plasmodium\t\r \u00a0falciparum\t\r \u00a0proteome\t\r \u00a0by\t\r \u00a0high-\u00ad\u2010accuracy\t\r \u00a0mass\t\r \u00a0spectrometry.\t\r \u00a0Nature\t\r \u00a0419,\t\r \u00a0537-\u00ad\u2010542\t\r \u00a0(2002).\t\r \u00a0364.\t\r \u00a0 Y.\t\r \u00a0Fang,\t\r \u00a0D.\t\r \u00a0P.\t\r \u00a0Robinson,\t\r \u00a0L.\t\r \u00a0J.\t\r \u00a0Foster,\t\r \u00a0Quantitative\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0proteome\t\r \u00a0coverage\t\r \u00a0and\t\r \u00a0recovery\t\r \u00a0rates\t\r \u00a0for\t\r \u00a0upstream\t\r \u00a0fractionation\t\r \u00a0methods\t\r \u00a0in\t\r \u00a0proteomics.\t\r \u00a0J\t\r \u00a0Proteome\t\r \u00a0Res\t\r \u00a09,\t\r \u00a01902-\u00ad\u20101912\t\r \u00a0(2010).\t\r \u00a0365.\t\r \u00a0 R.\t\r \u00a0J.\t\r \u00a0Slebos,\t\r \u00a0J.\t\r \u00a0W.\t\r \u00a0Brock,\t\r \u00a0N.\t\r \u00a0F.\t\r \u00a0Winters,\t\r \u00a0S.\t\r \u00a0R.\t\r \u00a0Stuart,\t\r \u00a0M.\t\r \u00a0A.\t\r \u00a0Martinez,\t\r \u00a0M.\t\r \u00a0Li,\t\r \u00a0M.\t\r \u00a0C.\t\r \u00a0Chambers,\t\r \u00a0L.\t\r \u00a0J.\t\r \u00a0Zimmerman,\t\r \u00a0A.\t\r \u00a0J.\t\r \u00a0Ham,\t\r \u00a0D.\t\r \u00a0L.\t\r \u00a0Tabb,\t\r \u00a0D.\t\r \u00a0C.\t\r \u00a0Liebler,\t\r \u00a0Evaluation\t\r \u00a0of\t\r \u00a0strong\t\r \u00a0cation\t\r \u00a0exchange\t\r \u00a0versus\t\r \u00a0isoelectric\t\r \u00a0focusing\t\r \u00a0of\t\r \u00a0peptides\t\r \u00a0for\t\r \u00a0multidimensional\t\r \u00a0liquid\t\r \u00a0chromatography-\u00ad\u2010tandem\t\r \u00a0mass\t\r \u00a0spectrometry.\t\r \u00a0J\t\r \u00a0 Proteome\t\r \u00a0Res\t\r \u00a07,\t\r \u00a05286-\u00ad\u20105294\t\r \u00a0(2008).\t\r \u00a0366.\t\r \u00a0 L.\t\r \u00a0Trinkle-\u00ad\u2010Mulcahy,\t\r \u00a0S.\t\r \u00a0Boulon,\t\r \u00a0Y.\t\r \u00a0W.\t\r \u00a0Lam,\t\r \u00a0R.\t\r \u00a0Urcia,\t\r \u00a0F.\t\r \u00a0M.\t\r \u00a0Boisvert,\t\r \u00a0F.\t\r \u00a0Vandermoere,\t\r \u00a0N.\t\r \u00a0A.\t\r \u00a0Morrice,\t\r \u00a0S.\t\r \u00a0Swift,\t\r \u00a0U.\t\r \u00a0Rothbauer,\t\r \u00a0H.\t\r \u00a0Leonhardt,\t\r \u00a0A.\t\r \u00a0Lamond,\t\r \u00a0Identifying\t\r \u00a0specific\t\r \u00a0protein\t\r \u00a0interaction\t\r \u00a0partners\t\r \u00a0using\t\r \u00a0quantitative\t\r \u00a0mass\t\r \u00a0spectrometry\t\r \u00a0and\t\r \u00a0bead\t\r \u00a0proteomes.\t\r \u00a0J\t\r \u00a0Cell\t\r \u00a0Biol\t\r \u00a0183,\t\r \u00a0223-\u00ad\u2010239\t\r \u00a0(2008).\t\r \u00a0367.\t\r \u00a0 L.\t\r \u00a0D.\t\r \u00a0Rogers,\t\r \u00a0L.\t\r \u00a0J.\t\r \u00a0Foster,\t\r \u00a0The\t\r \u00a0dynamic\t\r \u00a0phagosomal\t\r \u00a0proteome\t\r \u00a0and\t\r \u00a0the\t\r \u00a0contribution\t\r \u00a0of\t\r \u00a0the\t\r \u00a0endoplasmic\t\r \u00a0reticulum.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a0104,\t\r \u00a018520-\u00ad\u201018525\t\r \u00a0(2007).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 171\t\r \u00a0 368.\t\r \u00a0 B.\t\r \u00a0Cox,\t\r \u00a0A.\t\r \u00a0Emili,\t\r \u00a0Tissue\t\r \u00a0subcellular\t\r \u00a0fractionation\t\r \u00a0and\t\r \u00a0protein\t\r \u00a0extraction\t\r \u00a0for\t\r \u00a0use\t\r \u00a0in\t\r \u00a0mass-\u00ad\u2010spectrometry-\u00ad\u2010based\t\r \u00a0proteomics.\t\r \u00a0Nat\t\r \u00a0Protoc\t\r \u00a01,\t\r \u00a01872-\u00ad\u20101878\t\r \u00a0(2006).\t\r \u00a0369.\t\r \u00a0 N.\t\r \u00a0J.\t\r \u00a0Krogan,\t\r \u00a0G.\t\r \u00a0Cagney,\t\r \u00a0H.\t\r \u00a0Yu,\t\r \u00a0G.\t\r \u00a0Zhong,\t\r \u00a0X.\t\r \u00a0Guo,\t\r \u00a0A.\t\r \u00a0Ignatchenko,\t\r \u00a0J.\t\r \u00a0Li,\t\r \u00a0S.\t\r \u00a0Pu,\t\r \u00a0N.\t\r \u00a0Datta,\t\r \u00a0A.\t\r \u00a0P.\t\r \u00a0Tikuisis,\t\r \u00a0T.\t\r \u00a0Punna,\t\r \u00a0J.\t\r \u00a0M.\t\r \u00a0Peregrin-\u00ad\u2010Alvarez,\t\r \u00a0M.\t\r \u00a0Shales,\t\r \u00a0X.\t\r \u00a0Zhang,\t\r \u00a0M.\t\r \u00a0Davey,\t\r \u00a0M.\t\r \u00a0D.\t\r \u00a0Robinson,\t\r \u00a0A.\t\r \u00a0Paccanaro,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Bray,\t\r \u00a0A.\t\r \u00a0Sheung,\t\r \u00a0B.\t\r \u00a0Beattie,\t\r \u00a0D.\t\r \u00a0P.\t\r \u00a0Richards,\t\r \u00a0V.\t\r \u00a0Canadien,\t\r \u00a0A.\t\r \u00a0Lalev,\t\r \u00a0F.\t\r \u00a0Mena,\t\r \u00a0P.\t\r \u00a0Wong,\t\r \u00a0A.\t\r \u00a0Starostine,\t\r \u00a0M.\t\r \u00a0M.\t\r \u00a0Canete,\t\r \u00a0J.\t\r \u00a0Vlasblom,\t\r \u00a0S.\t\r \u00a0Wu,\t\r \u00a0C.\t\r \u00a0Orsi,\t\r \u00a0S.\t\r \u00a0R.\t\r \u00a0Collins,\t\r \u00a0S.\t\r \u00a0Chandran,\t\r \u00a0R.\t\r \u00a0Haw,\t\r \u00a0J.\t\r \u00a0J.\t\r \u00a0Rilstone,\t\r \u00a0K.\t\r \u00a0Gandi,\t\r \u00a0N.\t\r \u00a0J.\t\r \u00a0Thompson,\t\r \u00a0G.\t\r \u00a0Musso,\t\r \u00a0P.\t\r \u00a0St\t\r \u00a0Onge,\t\r \u00a0S.\t\r \u00a0Ghanny,\t\r \u00a0M.\t\r \u00a0H.\t\r \u00a0Lam,\t\r \u00a0G.\t\r \u00a0Butland,\t\r \u00a0A.\t\r \u00a0M.\t\r \u00a0Altaf-\u00ad\u2010Ul,\t\r \u00a0S.\t\r \u00a0Kanaya,\t\r \u00a0A.\t\r \u00a0Shilatifard,\t\r \u00a0E.\t\r \u00a0O'Shea,\t\r \u00a0J.\t\r \u00a0S.\t\r \u00a0Weissman,\t\r \u00a0C.\t\r \u00a0J.\t\r \u00a0Ingles,\t\r \u00a0T.\t\r \u00a0R.\t\r \u00a0Hughes,\t\r \u00a0J.\t\r \u00a0Parkinson,\t\r \u00a0M.\t\r \u00a0Gerstein,\t\r \u00a0S.\t\r \u00a0J.\t\r \u00a0Wodak,\t\r \u00a0A.\t\r \u00a0Emili,\t\r \u00a0J.\t\r \u00a0F.\t\r \u00a0Greenblatt,\t\r \u00a0Global\t\r \u00a0landscape\t\r \u00a0of\t\r \u00a0protein\t\r \u00a0complexes\t\r \u00a0in\t\r \u00a0the\t\r \u00a0yeast\t\r \u00a0Saccharomyces\t\r \u00a0cerevisiae.\t\r \u00a0Nature\t\r \u00a0440,\t\r \u00a0637-\u00ad\u2010643\t\r \u00a0(2006).\t\r \u00a0370.\t\r \u00a0 S.\t\r \u00a0C.\t\r \u00a0Kim,\t\r \u00a0R.\t\r \u00a0Sprung,\t\r \u00a0Y.\t\r \u00a0Chen,\t\r \u00a0Y.\t\r \u00a0Xu,\t\r \u00a0H.\t\r \u00a0Ball,\t\r \u00a0J.\t\r \u00a0Pei,\t\r \u00a0T.\t\r \u00a0Cheng,\t\r \u00a0Y.\t\r \u00a0Kho,\t\r \u00a0H.\t\r \u00a0Xiao,\t\r \u00a0L.\t\r \u00a0Xiao,\t\r \u00a0N.\t\r \u00a0V.\t\r \u00a0Grishin,\t\r \u00a0M.\t\r \u00a0White,\t\r \u00a0X.\t\r \u00a0J.\t\r \u00a0Yang,\t\r \u00a0Y.\t\r \u00a0Zhao,\t\r \u00a0Substrate\t\r \u00a0and\t\r \u00a0functional\t\r \u00a0diversity\t\r \u00a0of\t\r \u00a0lysine\t\r \u00a0acetylation\t\r \u00a0revealed\t\r \u00a0by\t\r \u00a0a\t\r \u00a0proteomics\t\r \u00a0survey.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a023,\t\r \u00a0607-\u00ad\u2010618\t\r \u00a0(2006).\t\r \u00a0371.\t\r \u00a0 S.\t\r \u00a0E.\t\r \u00a0Ong,\t\r \u00a0G.\t\r \u00a0Mittler,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Identifying\t\r \u00a0and\t\r \u00a0quantifying\t\r \u00a0in\t\r \u00a0vivo\t\r \u00a0methylation\t\r \u00a0sites\t\r \u00a0by\t\r \u00a0heavy\t\r \u00a0methyl\t\r \u00a0SILAC.\t\r \u00a0Nat\t\r \u00a0Methods\t\r \u00a01,\t\r \u00a0119-\u00ad\u2010126\t\r \u00a0(2004).\t\r \u00a0372.\t\r \u00a0 Y.\t\r \u00a0Oda,\t\r \u00a0T.\t\r \u00a0Nagasu,\t\r \u00a0B.\t\r \u00a0T.\t\r \u00a0Chait,\t\r \u00a0Enrichment\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0phosphorylated\t\r \u00a0proteins\t\r \u00a0as\t\r \u00a0a\t\r \u00a0tool\t\r \u00a0for\t\r \u00a0probing\t\r \u00a0the\t\r \u00a0phosphoproteome.\t\r \u00a0Nat\t\r \u00a0Biotechnol\t\r \u00a019,\t\r \u00a0379-\u00ad\u2010382\t\r \u00a0(2001).\t\r \u00a0373.\t\r \u00a0 L.\t\r \u00a0Wells,\t\r \u00a0K.\t\r \u00a0Vosseller,\t\r \u00a0R.\t\r \u00a0N.\t\r \u00a0Cole,\t\r \u00a0J.\t\r \u00a0M.\t\r \u00a0Cronshaw,\t\r \u00a0M.\t\r \u00a0J.\t\r \u00a0Matunis,\t\r \u00a0G.\t\r \u00a0W.\t\r \u00a0Hart,\t\r \u00a0Mapping\t\r \u00a0sites\t\r \u00a0of\t\r \u00a0O-\u00ad\u2010GlcNAc\t\r \u00a0modification\t\r \u00a0using\t\r \u00a0affinity\t\r \u00a0tags\t\r \u00a0for\t\r \u00a0serine\t\r \u00a0and\t\r \u00a0threonine\t\r \u00a0post-\u00ad\u2010translational\t\r \u00a0modifications.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Proteomics\t\r \u00a01,\t\r \u00a0791-\u00ad\u2010804\t\r \u00a0(2002).\t\r \u00a0374.\t\r \u00a0 F.\t\r \u00a0Elortza,\t\r \u00a0T.\t\r \u00a0S.\t\r \u00a0Nuhse,\t\r \u00a0L.\t\r \u00a0J.\t\r \u00a0Foster,\t\r \u00a0A.\t\r \u00a0Stensballe,\t\r \u00a0S.\t\r \u00a0C.\t\r \u00a0Peck,\t\r \u00a0O.\t\r \u00a0N.\t\r \u00a0Jensen,\t\r \u00a0Proteomic\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0glycosylphosphatidylinositol-\u00ad\u2010anchored\t\r \u00a0membrane\t\r \u00a0proteins.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Proteomics\t\r \u00a02,\t\r \u00a01261-\u00ad\u20101270\t\r \u00a0(2003).\t\r \u00a0375.\t\r \u00a0 S.\t\r \u00a0B.\t\r \u00a0Ficarro,\t\r \u00a0M.\t\r \u00a0L.\t\r \u00a0McCleland,\t\r \u00a0P.\t\r \u00a0T.\t\r \u00a0Stukenberg,\t\r \u00a0D.\t\r \u00a0J.\t\r \u00a0Burke,\t\r \u00a0M.\t\r \u00a0M.\t\r \u00a0Ross,\t\r \u00a0J.\t\r \u00a0Shabanowitz,\t\r \u00a0D.\t\r \u00a0F.\t\r \u00a0Hunt,\t\r \u00a0F.\t\r \u00a0M.\t\r \u00a0White,\t\r \u00a0Phosphoproteome\t\r \u00a0analysis\t\r \u00a0by\t\r \u00a0mass\t\r \u00a0spectrometry\t\r \u00a0and\t\r \u00a0its\t\r \u00a0application\t\r \u00a0to\t\r \u00a0Saccharomyces\t\r \u00a0cerevisiae.\t\r \u00a0Nat\t\r \u00a0Biotechnol\t\r \u00a0 20,\t\r \u00a0301-\u00ad\u2010305\t\r \u00a0(2002).\t\r \u00a0376.\t\r \u00a0 M.\t\r \u00a0R.\t\r \u00a0Larsen,\t\r \u00a0T.\t\r \u00a0E.\t\r \u00a0Thingholm,\t\r \u00a0O.\t\r \u00a0N.\t\r \u00a0Jensen,\t\r \u00a0P.\t\r \u00a0Roepstorff,\t\r \u00a0T.\t\r \u00a0J.\t\r \u00a0Jorgensen,\t\r \u00a0Highly\t\r \u00a0selective\t\r \u00a0enrichment\t\r \u00a0of\t\r \u00a0phosphorylated\t\r \u00a0peptides\t\r \u00a0from\t\r \u00a0peptide\t\r \u00a0mixtures\t\r \u00a0using\t\r \u00a0titanium\t\r \u00a0dioxide\t\r \u00a0microcolumns.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Proteomics\t\r \u00a04,\t\r \u00a0873-\u00ad\u2010886\t\r \u00a0(2005).\t\r \u00a0377.\t\r \u00a0 J.\t\r \u00a0Peng,\t\r \u00a0D.\t\r \u00a0Schwartz,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Elias,\t\r \u00a0C.\t\r \u00a0C.\t\r \u00a0Thoreen,\t\r \u00a0D.\t\r \u00a0Cheng,\t\r \u00a0G.\t\r \u00a0Marsischky,\t\r \u00a0J.\t\r \u00a0Roelofs,\t\r \u00a0D.\t\r \u00a0Finley,\t\r \u00a0S.\t\r \u00a0P.\t\r \u00a0Gygi,\t\r \u00a0A\t\r \u00a0proteomics\t\r \u00a0approach\t\r \u00a0to\t\r \u00a0understanding\t\r \u00a0protein\t\r \u00a0ubiquitination.\t\r \u00a0Nat\t\r \u00a0Biotechnol\t\r \u00a021,\t\r \u00a0921-\u00ad\u2010926\t\r \u00a0(2003).\t\r \u00a0378.\t\r \u00a0 O.\t\r \u00a0Kleifeld,\t\r \u00a0A.\t\r \u00a0Doucet,\t\r \u00a0U.\t\r \u00a0auf\t\r \u00a0dem\t\r \u00a0Keller,\t\r \u00a0A.\t\r \u00a0Prudova,\t\r \u00a0O.\t\r \u00a0Schilling,\t\r \u00a0R.\t\r \u00a0K.\t\r \u00a0Kainthan,\t\r \u00a0A.\t\r \u00a0E.\t\r \u00a0Starr,\t\r \u00a0L.\t\r \u00a0J.\t\r \u00a0Foster,\t\r \u00a0J.\t\r \u00a0N.\t\r \u00a0Kizhakkedathu,\t\r \u00a0C.\t\r \u00a0M.\t\r \u00a0Overall,\t\r \u00a0Isotopic\t\r \u00a0labeling\t\r \u00a0of\t\r \u00a0terminal\t\r \u00a0amines\t\r \u00a0in\t\r \u00a0complex\t\r \u00a0samples\t\r \u00a0identifies\t\r \u00a0protein\t\r \u00a0N-\u00ad\u2010termini\t\r \u00a0and\t\r \u00a0protease\t\r \u00a0cleavage\t\r \u00a0products.\t\r \u00a0Nat\t\r \u00a0Biotechnol\t\r \u00a028,\t\r \u00a0281-\u00ad\u2010288\t\r \u00a0(2010).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 172\t\r \u00a0 379.\t\r \u00a0 Y.\t\r \u00a0Zhao,\t\r \u00a0O.\t\r \u00a0N.\t\r \u00a0Jensen,\t\r \u00a0Modification-\u00ad\u2010specific\t\r \u00a0proteomics:\t\r \u00a0strategies\t\r \u00a0for\t\r \u00a0characterization\t\r \u00a0of\t\r \u00a0post-\u00ad\u2010translational\t\r \u00a0modifications\t\r \u00a0using\t\r \u00a0enrichment\t\r \u00a0techniques.\t\r \u00a0Proteomics\t\r \u00a09,\t\r \u00a04632-\u00ad\u20104641\t\r \u00a0(2009).\t\r \u00a0380.\t\r \u00a0 P.\t\r \u00a0Cohen,\t\r \u00a0The\t\r \u00a0role\t\r \u00a0of\t\r \u00a0protein\t\r \u00a0phosphorylation\t\r \u00a0in\t\r \u00a0human\t\r \u00a0health\t\r \u00a0and\t\r \u00a0disease.\t\r \u00a0The\t\r \u00a0Sir\t\r \u00a0Hans\t\r \u00a0Krebs\t\r \u00a0Medal\t\r \u00a0Lecture.\t\r \u00a0Eur\t\r \u00a0J\t\r \u00a0Biochem\t\r \u00a0268,\t\r \u00a05001-\u00ad\u20105010\t\r \u00a0(2001).\t\r \u00a0381.\t\r \u00a0 B.\t\r \u00a0Macek,\t\r \u00a0I.\t\r \u00a0Mijakovic,\t\r \u00a0J.\t\r \u00a0V.\t\r \u00a0Olsen,\t\r \u00a0F.\t\r \u00a0Gnad,\t\r \u00a0C.\t\r \u00a0Kumar,\t\r \u00a0P.\t\r \u00a0R.\t\r \u00a0Jensen,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0The\t\r \u00a0serine\/threonine\/tyrosine\t\r \u00a0phosphoproteome\t\r \u00a0of\t\r \u00a0the\t\r \u00a0model\t\r \u00a0bacterium\t\r \u00a0Bacillus\t\r \u00a0subtilis.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Proteomics\t\r \u00a06,\t\r \u00a0697-\u00ad\u2010707\t\r \u00a0(2007).\t\r \u00a0382.\t\r \u00a0 B.\t\r \u00a0Macek,\t\r \u00a0F.\t\r \u00a0Gnad,\t\r \u00a0B.\t\r \u00a0Soufi,\t\r \u00a0C.\t\r \u00a0Kumar,\t\r \u00a0J.\t\r \u00a0V.\t\r \u00a0Olsen,\t\r \u00a0I.\t\r \u00a0Mijakovic,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Phosphoproteome\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0E.\t\r \u00a0coli\t\r \u00a0reveals\t\r \u00a0evolutionary\t\r \u00a0conservation\t\r \u00a0of\t\r \u00a0bacterial\t\r \u00a0Ser\/Thr\/Tyr\t\r \u00a0phosphorylation.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Proteomics\t\r \u00a07,\t\r \u00a0299-\u00ad\u2010307\t\r \u00a0(2008).\t\r \u00a0383.\t\r \u00a0 S.\t\r \u00a0Voisin,\t\r \u00a0D.\t\r \u00a0C.\t\r \u00a0Watson,\t\r \u00a0L.\t\r \u00a0Tessier,\t\r \u00a0W.\t\r \u00a0Ding,\t\r \u00a0S.\t\r \u00a0Foote,\t\r \u00a0S.\t\r \u00a0Bhatia,\t\r \u00a0J.\t\r \u00a0F.\t\r \u00a0Kelly,\t\r \u00a0N.\t\r \u00a0M.\t\r \u00a0Young,\t\r \u00a0The\t\r \u00a0cytoplasmic\t\r \u00a0phosphoproteome\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Gram-\u00ad\u2010negative\t\r \u00a0bacterium\t\r \u00a0Campylobacter\t\r \u00a0jejuni:\t\r \u00a0evidence\t\r \u00a0for\t\r \u00a0modification\t\r \u00a0by\t\r \u00a0unidentified\t\r \u00a0protein\t\r \u00a0kinases.\t\r \u00a0Proteomics\t\r \u00a07,\t\r \u00a04338-\u00ad\u20104348\t\r \u00a0(2007).\t\r \u00a0384.\t\r \u00a0 B.\t\r \u00a0Soufi,\t\r \u00a0F.\t\r \u00a0Gnad,\t\r \u00a0P.\t\r \u00a0R.\t\r \u00a0Jensen,\t\r \u00a0D.\t\r \u00a0Petranovic,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0I.\t\r \u00a0Mijakovic,\t\r \u00a0B.\t\r \u00a0Macek,\t\r \u00a0The\t\r \u00a0Ser\/Thr\/Tyr\t\r \u00a0phosphoproteome\t\r \u00a0of\t\r \u00a0Lactococcus\t\r \u00a0lactis\t\r \u00a0IL1403\t\r \u00a0reveals\t\r \u00a0multiply\t\r \u00a0phosphorylated\t\r \u00a0proteins.\t\r \u00a0Proteomics\t\r \u00a08,\t\r \u00a03486-\u00ad\u20103493\t\r \u00a0(2008).\t\r \u00a0385.\t\r \u00a0 A.\t\r \u00a0Alonso,\t\r \u00a0J.\t\r \u00a0Sasin,\t\r \u00a0N.\t\r \u00a0Bottini,\t\r \u00a0I.\t\r \u00a0Friedberg,\t\r \u00a0I.\t\r \u00a0Friedberg,\t\r \u00a0A.\t\r \u00a0Osterman,\t\r \u00a0A.\t\r \u00a0Godzik,\t\r \u00a0T.\t\r \u00a0Hunter,\t\r \u00a0J.\t\r \u00a0Dixon,\t\r \u00a0T.\t\r \u00a0Mustelin,\t\r \u00a0Protein\t\r \u00a0tyrosine\t\r \u00a0phosphatases\t\r \u00a0in\t\r \u00a0the\t\r \u00a0human\t\r \u00a0genome.\t\r \u00a0Cell\t\r \u00a0117,\t\r \u00a0699-\u00ad\u2010711\t\r \u00a0(2004).\t\r \u00a0386.\t\r \u00a0 A.\t\r \u00a0Remenyi,\t\r \u00a0M.\t\r \u00a0C.\t\r \u00a0Good,\t\r \u00a0W.\t\r \u00a0A.\t\r \u00a0Lim,\t\r \u00a0Docking\t\r \u00a0interactions\t\r \u00a0in\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a0and\t\r \u00a0phosphatase\t\r \u00a0networks.\t\r \u00a0Curr\t\r \u00a0Opin\t\r \u00a0Struct\t\r \u00a0Biol\t\r \u00a016,\t\r \u00a0676-\u00ad\u2010685\t\r \u00a0(2006).\t\r \u00a0387.\t\r \u00a0 C.\t\r \u00a0Pan,\t\r \u00a0F.\t\r \u00a0Gnad,\t\r \u00a0J.\t\r \u00a0V.\t\r \u00a0Olsen,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Quantitative\t\r \u00a0phosphoproteome\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0a\t\r \u00a0mouse\t\r \u00a0liver\t\r \u00a0cell\t\r \u00a0line\t\r \u00a0reveals\t\r \u00a0specificity\t\r \u00a0of\t\r \u00a0phosphatase\t\r \u00a0inhibitors.\t\r \u00a0 Proteomics\t\r \u00a08,\t\r \u00a04534-\u00ad\u20104546\t\r \u00a0(2008).\t\r \u00a0388.\t\r \u00a0 T.\t\r \u00a0E.\t\r \u00a0Thingholm,\t\r \u00a0M.\t\r \u00a0R.\t\r \u00a0Larsen,\t\r \u00a0C.\t\r \u00a0R.\t\r \u00a0Ingrell,\t\r \u00a0M.\t\r \u00a0Kassem,\t\r \u00a0O.\t\r \u00a0N.\t\r \u00a0Jensen,\t\r \u00a0TiO(2)-\u00ad\u2010based\t\r \u00a0phosphoproteomic\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0the\t\r \u00a0plasma\t\r \u00a0membrane\t\r \u00a0and\t\r \u00a0the\t\r \u00a0effects\t\r \u00a0of\t\r \u00a0phosphatase\t\r \u00a0inhibitor\t\r \u00a0treatment.\t\r \u00a0J\t\r \u00a0Proteome\t\r \u00a0Res\t\r \u00a07,\t\r \u00a03304-\u00ad\u20103313\t\r \u00a0(2008).\t\r \u00a0389.\t\r \u00a0 S.\t\r \u00a0A.\t\r \u00a0Beausoleil,\t\r \u00a0M.\t\r \u00a0Jedrychowski,\t\r \u00a0D.\t\r \u00a0Schwartz,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Elias,\t\r \u00a0J.\t\r \u00a0Villen,\t\r \u00a0J.\t\r \u00a0Li,\t\r \u00a0M.\t\r \u00a0A.\t\r \u00a0Cohn,\t\r \u00a0L.\t\r \u00a0C.\t\r \u00a0Cantley,\t\r \u00a0S.\t\r \u00a0P.\t\r \u00a0Gygi,\t\r \u00a0Large-\u00ad\u2010scale\t\r \u00a0characterization\t\r \u00a0of\t\r \u00a0HeLa\t\r \u00a0cell\t\r \u00a0nuclear\t\r \u00a0phosphoproteins.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a0101,\t\r \u00a012130-\u00ad\u201012135\t\r \u00a0(2004).\t\r \u00a0390.\t\r \u00a0 A.\t\r \u00a0Gruhler,\t\r \u00a0J.\t\r \u00a0V.\t\r \u00a0Olsen,\t\r \u00a0S.\t\r \u00a0Mohammed,\t\r \u00a0P.\t\r \u00a0Mortensen,\t\r \u00a0N.\t\r \u00a0J.\t\r \u00a0Faergeman,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0O.\t\r \u00a0N.\t\r \u00a0Jensen,\t\r \u00a0Quantitative\t\r \u00a0phosphoproteomics\t\r \u00a0applied\t\r \u00a0to\t\r \u00a0the\t\r \u00a0yeast\t\r \u00a0pheromone\t\r \u00a0signaling\t\r \u00a0pathway.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Proteomics\t\r \u00a04,\t\r \u00a0310-\u00ad\u2010327\t\r \u00a0(2005).\t\r \u00a0391.\t\r \u00a0 T.\t\r \u00a0S.\t\r \u00a0Nuhse,\t\r \u00a0A.\t\r \u00a0Stensballe,\t\r \u00a0O.\t\r \u00a0N.\t\r \u00a0Jensen,\t\r \u00a0S.\t\r \u00a0C.\t\r \u00a0Peck,\t\r \u00a0Large-\u00ad\u2010scale\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0in\t\r \u00a0vivo\t\r \u00a0phosphorylated\t\r \u00a0membrane\t\r \u00a0proteins\t\r \u00a0by\t\r \u00a0immobilized\t\r \u00a0metal\t\r \u00a0ion\t\r \u00a0affinity\t\r \u00a0chromatography\t\r \u00a0and\t\r \u00a0mass\t\r \u00a0spectrometry.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Proteomics\t\r \u00a02,\t\r \u00a01234-\u00ad\u20101243\t\r \u00a0(2003).\t\r \u00a0392.\t\r \u00a0 J.\t\r \u00a0Dai,\t\r \u00a0L.\t\r \u00a0S.\t\r \u00a0Wang,\t\r \u00a0Y.\t\r \u00a0B.\t\r \u00a0Wu,\t\r \u00a0Q.\t\r \u00a0H.\t\r \u00a0Sheng,\t\r \u00a0J.\t\r \u00a0R.\t\r \u00a0Wu,\t\r \u00a0C.\t\r \u00a0H.\t\r \u00a0Shieh,\t\r \u00a0R.\t\r \u00a0Zeng,\t\r \u00a0Fully\t\r \u00a0automatic\t\r \u00a0separation\t\r \u00a0and\t\r \u00a0identification\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0by\t\r \u00a0continuous\t\r \u00a0pH-\u00ad\u2010gradient\t\r \u00a0anion\t\r \u00a0exchange\t\r \u00a0online\t\r \u00a0coupled\t\r \u00a0with\t\r \u00a0reversed-\u00ad\u2010phase\t\r \u00a0liquid\t\r \u00a0chromatography\t\r \u00a0mass\t\r \u00a0spectrometry.\t\r \u00a0J\t\r \u00a0Proteome\t\r \u00a0Res\t\r \u00a08,\t\r \u00a0133-\u00ad\u2010141\t\r \u00a0(2009).\t\r \u00a0393.\t\r \u00a0 T.\t\r \u00a0E.\t\r \u00a0Thingholm,\t\r \u00a0O.\t\r \u00a0N.\t\r \u00a0Jensen,\t\r \u00a0M.\t\r \u00a0R.\t\r \u00a0Larsen,\t\r \u00a0Analytical\t\r \u00a0strategies\t\r \u00a0for\t\r \u00a0phosphoproteomics.\t\r \u00a0Proteomics\t\r \u00a09,\t\r \u00a01451-\u00ad\u20101468\t\r \u00a0(2009).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 173\t\r \u00a0 394.\t\r \u00a0 A.\t\r \u00a0J.\t\r \u00a0Alpert,\t\r \u00a0Hydrophilic-\u00ad\u2010interaction\t\r \u00a0chromatography\t\r \u00a0for\t\r \u00a0the\t\r \u00a0separation\t\r \u00a0of\t\r \u00a0peptides,\t\r \u00a0nucleic\t\r \u00a0acids\t\r \u00a0and\t\r \u00a0other\t\r \u00a0polar\t\r \u00a0compounds.\t\r \u00a0J\t\r \u00a0Chromatogr\t\r \u00a0499,\t\r \u00a0177-\u00ad\u2010196\t\r \u00a0(1990).\t\r \u00a0395.\t\r \u00a0 D.\t\r \u00a0E.\t\r \u00a0McNulty,\t\r \u00a0R.\t\r \u00a0S.\t\r \u00a0Annan,\t\r \u00a0Hydrophilic\t\r \u00a0interaction\t\r \u00a0chromatography\t\r \u00a0reduces\t\r \u00a0the\t\r \u00a0complexity\t\r \u00a0of\t\r \u00a0the\t\r \u00a0phosphoproteome\t\r \u00a0and\t\r \u00a0improves\t\r \u00a0global\t\r \u00a0phosphopeptide\t\r \u00a0isolation\t\r \u00a0and\t\r \u00a0detection.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Proteomics\t\r \u00a07,\t\r \u00a0971-\u00ad\u2010980\t\r \u00a0(2008).\t\r \u00a0396.\t\r \u00a0 A.\t\r \u00a0J.\t\r \u00a0Alpert,\t\r \u00a0Electrostatic\t\r \u00a0repulsion\t\r \u00a0hydrophilic\t\r \u00a0interaction\t\r \u00a0chromatography\t\r \u00a0for\t\r \u00a0isocratic\t\r \u00a0separation\t\r \u00a0of\t\r \u00a0charged\t\r \u00a0solutes\t\r \u00a0and\t\r \u00a0selective\t\r \u00a0isolation\t\r \u00a0of\t\r \u00a0phosphopeptides.\t\r \u00a0Anal\t\r \u00a0Chem\t\r \u00a080,\t\r \u00a062-\u00ad\u201076\t\r \u00a0(2008).\t\r \u00a0397.\t\r \u00a0 C.\t\r \u00a0S.\t\r \u00a0Gan,\t\r \u00a0T.\t\r \u00a0Guo,\t\r \u00a0H.\t\r \u00a0Zhang,\t\r \u00a0S.\t\r \u00a0K.\t\r \u00a0Lim,\t\r \u00a0S.\t\r \u00a0K.\t\r \u00a0Sze,\t\r \u00a0A\t\r \u00a0comparative\t\r \u00a0study\t\r \u00a0of\t\r \u00a0electrostatic\t\r \u00a0repulsion-\u00ad\u2010hydrophilic\t\r \u00a0interaction\t\r \u00a0chromatography\t\r \u00a0(ERLIC)\t\r \u00a0versus\t\r \u00a0SCX-\u00ad\u2010IMAC-\u00ad\u2010based\t\r \u00a0methods\t\r \u00a0for\t\r \u00a0phosphopeptide\t\r \u00a0isolation\/enrichment.\t\r \u00a0J\t\r \u00a0 Proteome\t\r \u00a0Res\t\r \u00a07,\t\r \u00a04869-\u00ad\u20104877\t\r \u00a0(2008).\t\r \u00a0398.\t\r \u00a0 X.\t\r \u00a0Zhang,\t\r \u00a0J.\t\r \u00a0Ye,\t\r \u00a0O.\t\r \u00a0N.\t\r \u00a0Jensen,\t\r \u00a0P.\t\r \u00a0Roepstorff,\t\r \u00a0Highly\t\r \u00a0Efficient\t\r \u00a0Phosphopeptide\t\r \u00a0Enrichment\t\r \u00a0by\t\r \u00a0Calcium\t\r \u00a0Phosphate\t\r \u00a0Precipitation\t\r \u00a0Combined\t\r \u00a0with\t\r \u00a0Subsequent\t\r \u00a0IMAC\t\r \u00a0Enrichment.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Proteomics\t\r \u00a06,\t\r \u00a02032-\u00ad\u20102042\t\r \u00a0(2007).\t\r \u00a0399.\t\r \u00a0 C.\t\r \u00a0I.\t\r \u00a0Ruse,\t\r \u00a0D.\t\r \u00a0B.\t\r \u00a0McClatchy,\t\r \u00a0B.\t\r \u00a0Lu,\t\r \u00a0D.\t\r \u00a0Cociorva,\t\r \u00a0A.\t\r \u00a0Motoyama,\t\r \u00a0S.\t\r \u00a0K.\t\r \u00a0Park,\t\r \u00a0J.\t\r \u00a0R.\t\r \u00a0Yates,\t\r \u00a03rd,\t\r \u00a0Motif-\u00ad\u2010specific\t\r \u00a0sampling\t\r \u00a0of\t\r \u00a0phosphoproteomes.\t\r \u00a0J\t\r \u00a0Proteome\t\r \u00a0Res\t\r \u00a07,\t\r \u00a02140-\u00ad\u20102150\t\r \u00a0(2008).\t\r \u00a0400.\t\r \u00a0 G.\t\r \u00a0Maccarrone,\t\r \u00a0N.\t\r \u00a0Kolb,\t\r \u00a0L.\t\r \u00a0Teplytska,\t\r \u00a0I.\t\r \u00a0Birg,\t\r \u00a0R.\t\r \u00a0Zollinger,\t\r \u00a0F.\t\r \u00a0Holsboer,\t\r \u00a0C.\t\r \u00a0W.\t\r \u00a0Turck,\t\r \u00a0Phosphopeptide\t\r \u00a0enrichment\t\r \u00a0by\t\r \u00a0IEF.\t\r \u00a0Electrophoresis\t\r \u00a027,\t\r \u00a04585-\u00ad\u20104595\t\r \u00a0(2006).\t\r \u00a0401.\t\r \u00a0 C.\t\r \u00a0W.\t\r \u00a0Hung,\t\r \u00a0D.\t\r \u00a0Kubler,\t\r \u00a0W.\t\r \u00a0D.\t\r \u00a0Lehmann,\t\r \u00a0pI-\u00ad\u2010based\t\r \u00a0phosphopeptide\t\r \u00a0enrichment\t\r \u00a0combined\t\r \u00a0with\t\r \u00a0nanoESI-\u00ad\u2010MS.\t\r \u00a0Electrophoresis\t\r \u00a028,\t\r \u00a02044-\u00ad\u20102052\t\r \u00a0(2007).\t\r \u00a0402.\t\r \u00a0 C.\t\r \u00a0F.\t\r \u00a0Xu,\t\r \u00a0H.\t\r \u00a0Wang,\t\r \u00a0D.\t\r \u00a0Li,\t\r \u00a0X.\t\r \u00a0P.\t\r \u00a0Kong,\t\r \u00a0T.\t\r \u00a0A.\t\r \u00a0Neubert,\t\r \u00a0Selective\t\r \u00a0enrichment\t\r \u00a0and\t\r \u00a0fractionation\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0from\t\r \u00a0peptide\t\r \u00a0mixtures\t\r \u00a0by\t\r \u00a0isoelectric\t\r \u00a0focusing\t\r \u00a0after\t\r \u00a0methyl\t\r \u00a0esterification.\t\r \u00a0Anal\t\r \u00a0Chem\t\r \u00a079,\t\r \u00a02007-\u00ad\u20102014\t\r \u00a0(2007).\t\r \u00a0403.\t\r \u00a0 B.\t\r \u00a0Blagoev,\t\r \u00a0S.\t\r \u00a0E.\t\r \u00a0Ong,\t\r \u00a0I.\t\r \u00a0Kratchmarova,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Temporal\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0phosphotyrosine-\u00ad\u2010dependent\t\r \u00a0signaling\t\r \u00a0networks\t\r \u00a0by\t\r \u00a0quantitative\t\r \u00a0proteomics.\t\r \u00a0 Nat.\t\r \u00a0Biotechnol.\t\r \u00a022,\t\r \u00a01139-\u00ad\u20101145\t\r \u00a0(2004).\t\r \u00a0404.\t\r \u00a0 M.\t\r \u00a0Kr\u00fcger,\t\r \u00a0I.\t\r \u00a0Kratchmarova,\t\r \u00a0B.\t\r \u00a0Blagoev,\t\r \u00a0Y.\t\r \u00a0H.\t\r \u00a0Tseng,\t\r \u00a0C.\t\r \u00a0R.\t\r \u00a0Kahn,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Dissection\t\r \u00a0of\t\r \u00a0the\t\r \u00a0insulin\t\r \u00a0signaling\t\r \u00a0pathway\t\r \u00a0via\t\r \u00a0quantitative\t\r \u00a0phosphoproteomics.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a0105,\t\r \u00a02451-\u00ad\u20102456\t\r \u00a0(2008).\t\r \u00a0405.\t\r \u00a0 S.\t\r \u00a0Matsuoka,\t\r \u00a0B.\t\r \u00a0A.\t\r \u00a0Ballif,\t\r \u00a0A.\t\r \u00a0Smogorzewska,\t\r \u00a0E.\t\r \u00a0R.\t\r \u00a0McDonald,\t\r \u00a03rd,\t\r \u00a0K.\t\r \u00a0E.\t\r \u00a0Hurov,\t\r \u00a0J.\t\r \u00a0Luo,\t\r \u00a0C.\t\r \u00a0E.\t\r \u00a0Bakalarski,\t\r \u00a0Z.\t\r \u00a0Zhao,\t\r \u00a0N.\t\r \u00a0Solimini,\t\r \u00a0Y.\t\r \u00a0Lerenthal,\t\r \u00a0Y.\t\r \u00a0Shiloh,\t\r \u00a0S.\t\r \u00a0P.\t\r \u00a0Gygi,\t\r \u00a0S.\t\r \u00a0J.\t\r \u00a0Elledge,\t\r \u00a0ATM\t\r \u00a0and\t\r \u00a0ATR\t\r \u00a0substrate\t\r \u00a0analysis\t\r \u00a0reveals\t\r \u00a0extensive\t\r \u00a0protein\t\r \u00a0networks\t\r \u00a0responsive\t\r \u00a0to\t\r \u00a0DNA\t\r \u00a0damage.\t\r \u00a0Science\t\r \u00a0316,\t\r \u00a01160-\u00ad\u20101166\t\r \u00a0(2007).\t\r \u00a0406.\t\r \u00a0 L.\t\r \u00a0Andersson,\t\r \u00a0J.\t\r \u00a0Porath,\t\r \u00a0Isolation\t\r \u00a0of\t\r \u00a0phosphoproteins\t\r \u00a0by\t\r \u00a0immobilized\t\r \u00a0metal\t\r \u00a0(Fe3+)\t\r \u00a0affinity\t\r \u00a0chromatography.\t\r \u00a0Anal\t\r \u00a0Biochem\t\r \u00a0154,\t\r \u00a0250-\u00ad\u2010254\t\r \u00a0(1986).\t\r \u00a0407.\t\r \u00a0 P.\t\r \u00a0Scanff,\t\r \u00a0M.\t\r \u00a0Yvon,\t\r \u00a0J.\t\r \u00a0P.\t\r \u00a0Pelissier,\t\r \u00a0Immobilized\t\r \u00a0Fe3+\t\r \u00a0affinity\t\r \u00a0chromatographic\t\r \u00a0isolation\t\r \u00a0of\t\r \u00a0phosphopeptides.\t\r \u00a0J\t\r \u00a0Chromatogr\t\r \u00a0539,\t\r \u00a0425-\u00ad\u2010432\t\r \u00a0(1991).\t\r \u00a0408.\t\r \u00a0 J.\t\r \u00a0A.\t\r \u00a0Karty,\t\r \u00a0J.\t\r \u00a0P.\t\r \u00a0Reilly,\t\r \u00a0Deamidation\t\r \u00a0as\t\r \u00a0a\t\r \u00a0consequence\t\r \u00a0of\t\r \u00a0beta-\u00ad\u2010elimination\t\r \u00a0of\t\r \u00a0phosphopeptides.\t\r \u00a0Anal\t\r \u00a0Chem\t\r \u00a077,\t\r \u00a04673-\u00ad\u20104676\t\r \u00a0(2005).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 174\t\r \u00a0 409.\t\r \u00a0 R.\t\r \u00a0J.\t\r \u00a0Seward,\t\r \u00a0Perlman,\t\r \u00a0D.\t\r \u00a0H.,\t\r \u00a0Berg,\t\r \u00a0E.\t\r \u00a0A.,\t\r \u00a0Hu,\t\r \u00a0J.,\t\r \u00a0Costello,\t\r \u00a0C.\t\r \u00a0E.,\t\r \u00a0paper\t\r \u00a0presented\t\r \u00a0at\t\r \u00a0the\t\r \u00a052nd\t\r \u00a0ASMS\t\r \u00a0Conference\t\r \u00a0on\t\r \u00a0Mass\t\r \u00a0Spectrometry\t\r \u00a0and\t\r \u00a0Allied\t\r \u00a0Topics,\t\r \u00a0Nashville,\t\r \u00a0USA,\t\r \u00a0May\t\r \u00a023-\u00ad\u201027\t\r \u00a02004.\t\r \u00a0410.\t\r \u00a0 R.\t\r \u00a0J.\t\r \u00a0Seward,\t\r \u00a0P.\t\r \u00a0D.\t\r \u00a0von\t\r \u00a0Haller,\t\r \u00a0R.\t\r \u00a0Aebersold,\t\r \u00a0B.\t\r \u00a0T.\t\r \u00a0Huber,\t\r \u00a0Phosphorylation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0pro-\u00ad\u2010apoptotic\t\r \u00a0protein\t\r \u00a0Bim\t\r \u00a0in\t\r \u00a0lymphocytes\t\r \u00a0is\t\r \u00a0associated\t\r \u00a0with\t\r \u00a0protection\t\r \u00a0from\t\r \u00a0apoptosis.\t\r \u00a0Mol\t\r \u00a0Immunol\t\r \u00a039,\t\r \u00a0983-\u00ad\u2010993\t\r \u00a0(2003).\t\r \u00a0411.\t\r \u00a0 M.\t\r \u00a0W.\t\r \u00a0Pinkse,\t\r \u00a0P.\t\r \u00a0M.\t\r \u00a0Uitto,\t\r \u00a0M.\t\r \u00a0J.\t\r \u00a0Hilhorst,\t\r \u00a0B.\t\r \u00a0Ooms,\t\r \u00a0A.\t\r \u00a0J.\t\r \u00a0Heck,\t\r \u00a0Selective\t\r \u00a0isolation\t\r \u00a0at\t\r \u00a0the\t\r \u00a0femtomole\t\r \u00a0level\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0from\t\r \u00a0proteolytic\t\r \u00a0digests\t\r \u00a0using\t\r \u00a02D-\u00ad\u2010NanoLC-\u00ad\u2010ESI-\u00ad\u2010MS\/MS\t\r \u00a0and\t\r \u00a0titanium\t\r \u00a0oxide\t\r \u00a0precolumns.\t\r \u00a0Anal\t\r \u00a0Chem\t\r \u00a076,\t\r \u00a03935-\u00ad\u20103943\t\r \u00a0(2004).\t\r \u00a0412.\t\r \u00a0 N.\t\r \u00a0Sugiyama,\t\r \u00a0T.\t\r \u00a0Masuda,\t\r \u00a0K.\t\r \u00a0Shinoda,\t\r \u00a0A.\t\r \u00a0Nakamura,\t\r \u00a0M.\t\r \u00a0Tomita,\t\r \u00a0Y.\t\r \u00a0Ishihama,\t\r \u00a0Phosphopeptide\t\r \u00a0enrichment\t\r \u00a0by\t\r \u00a0aliphatic\t\r \u00a0hydroxy\t\r \u00a0acid-\u00ad\u2010modified\t\r \u00a0metal\t\r \u00a0oxide\t\r \u00a0chromatography\t\r \u00a0for\t\r \u00a0nano-\u00ad\u2010LC-\u00ad\u2010MS\/MS\t\r \u00a0in\t\r \u00a0proteomics\t\r \u00a0applications.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0 Proteomics\t\r \u00a06,\t\r \u00a01103-\u00ad\u20101109\t\r \u00a0(2007).\t\r \u00a0413.\t\r \u00a0 H.\t\r \u00a0K.\t\r \u00a0Kweon,\t\r \u00a0K.\t\r \u00a0Hakansson,\t\r \u00a0Selective\t\r \u00a0zirconium\t\r \u00a0dioxide-\u00ad\u2010based\t\r \u00a0enrichment\t\r \u00a0of\t\r \u00a0phosphorylated\t\r \u00a0peptides\t\r \u00a0for\t\r \u00a0mass\t\r \u00a0spectrometric\t\r \u00a0analysis.\t\r \u00a0Anal\t\r \u00a0Chem\t\r \u00a078,\t\r \u00a01743-\u00ad\u20101749\t\r \u00a0(2006).\t\r \u00a0414.\t\r \u00a0 Y.\t\r \u00a0Kyono,\t\r \u00a0N.\t\r \u00a0Sugiyama,\t\r \u00a0K.\t\r \u00a0Imami,\t\r \u00a0M.\t\r \u00a0Tomita,\t\r \u00a0Y.\t\r \u00a0Ishihama,\t\r \u00a0Successive\t\r \u00a0and\t\r \u00a0selective\t\r \u00a0release\t\r \u00a0of\t\r \u00a0phosphorylated\t\r \u00a0peptides\t\r \u00a0captured\t\r \u00a0by\t\r \u00a0hydroxy\t\r \u00a0acid-\u00ad\u2010modified\t\r \u00a0metal\t\r \u00a0oxide\t\r \u00a0chromatography.\t\r \u00a0J\t\r \u00a0Proteome\t\r \u00a0Res\t\r \u00a07,\t\r \u00a04585-\u00ad\u20104593\t\r \u00a0(2008).\t\r \u00a0415.\t\r \u00a0 T.\t\r \u00a0E.\t\r \u00a0Thingholm,\t\r \u00a0O.\t\r \u00a0N.\t\r \u00a0Jensen,\t\r \u00a0P.\t\r \u00a0J.\t\r \u00a0Robinson,\t\r \u00a0M.\t\r \u00a0R.\t\r \u00a0Larsen,\t\r \u00a0SIMAC\t\r \u00a0(sequential\t\r \u00a0elution\t\r \u00a0from\t\r \u00a0IMAC),\t\r \u00a0a\t\r \u00a0phosphoproteomics\t\r \u00a0strategy\t\r \u00a0for\t\r \u00a0the\t\r \u00a0rapid\t\r \u00a0separation\t\r \u00a0of\t\r \u00a0monophosphorylated\t\r \u00a0from\t\r \u00a0multiply\t\r \u00a0phosphorylated\t\r \u00a0peptides.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0 Proteomics\t\r \u00a07,\t\r \u00a0661-\u00ad\u2010671\t\r \u00a0(2008).\t\r \u00a0416.\t\r \u00a0 D.\t\r \u00a0T.\t\r \u00a0McLachlin,\t\r \u00a0B.\t\r \u00a0T.\t\r \u00a0Chait,\t\r \u00a0Improved\t\r \u00a0beta-\u00ad\u2010elimination-\u00ad\u2010based\t\r \u00a0affinity\t\r \u00a0purification\t\r \u00a0strategy\t\r \u00a0for\t\r \u00a0enrichment\t\r \u00a0of\t\r \u00a0phosphopeptides.\t\r \u00a0Anal\t\r \u00a0Chem\t\r \u00a075,\t\r \u00a06826-\u00ad\u20106836\t\r \u00a0(2003).\t\r \u00a0417.\t\r \u00a0 Z.\t\r \u00a0A.\t\r \u00a0Knight,\t\r \u00a0B.\t\r \u00a0Schilling,\t\r \u00a0R.\t\r \u00a0H.\t\r \u00a0Row,\t\r \u00a0D.\t\r \u00a0M.\t\r \u00a0Kenski,\t\r \u00a0B.\t\r \u00a0W.\t\r \u00a0Gibson,\t\r \u00a0K.\t\r \u00a0M.\t\r \u00a0Shokat,\t\r \u00a0Phosphospecific\t\r \u00a0proteolysis\t\r \u00a0for\t\r \u00a0mapping\t\r \u00a0sites\t\r \u00a0of\t\r \u00a0protein\t\r \u00a0phosphorylation.\t\r \u00a0 Nat\t\r \u00a0Biotechnol\t\r \u00a021,\t\r \u00a01047-\u00ad\u20101054\t\r \u00a0(2003).\t\r \u00a0418.\t\r \u00a0 N.\t\r \u00a0Dephoure,\t\r \u00a0C.\t\r \u00a0Zhou,\t\r \u00a0J.\t\r \u00a0Villen,\t\r \u00a0S.\t\r \u00a0A.\t\r \u00a0Beausoleil,\t\r \u00a0C.\t\r \u00a0E.\t\r \u00a0Bakalarski,\t\r \u00a0S.\t\r \u00a0J.\t\r \u00a0Elledge,\t\r \u00a0S.\t\r \u00a0P.\t\r \u00a0Gygi,\t\r \u00a0A\t\r \u00a0quantitative\t\r \u00a0atlas\t\r \u00a0of\t\r \u00a0mitotic\t\r \u00a0phosphorylation.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0 A\t\r \u00a0105,\t\r \u00a010762-\u00ad\u201010767\t\r \u00a0(2008).\t\r \u00a0419.\t\r \u00a0 J.\t\r \u00a0P.\t\r \u00a0DeGnore,\t\r \u00a0J.\t\r \u00a0Qin,\t\r \u00a0Fragmentation\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0in\t\r \u00a0an\t\r \u00a0ion\t\r \u00a0trap\t\r \u00a0mass\t\r \u00a0spectrometer.\t\r \u00a0J\t\r \u00a0Am\t\r \u00a0Soc\t\r \u00a0Mass\t\r \u00a0Spectrom\t\r \u00a09,\t\r \u00a01175-\u00ad\u20101188\t\r \u00a0(1998).\t\r \u00a0420.\t\r \u00a0 M.\t\r \u00a0J.\t\r \u00a0Schroeder,\t\r \u00a0J.\t\r \u00a0Shabanowitz,\t\r \u00a0J.\t\r \u00a0C.\t\r \u00a0Schwartz,\t\r \u00a0D.\t\r \u00a0F.\t\r \u00a0Hunt,\t\r \u00a0J.\t\r \u00a0J.\t\r \u00a0Coon,\t\r \u00a0A\t\r \u00a0neutral\t\r \u00a0loss\t\r \u00a0activation\t\r \u00a0method\t\r \u00a0for\t\r \u00a0improved\t\r \u00a0phosphopeptide\t\r \u00a0sequence\t\r \u00a0analysis\t\r \u00a0by\t\r \u00a0quadrupole\t\r \u00a0ion\t\r \u00a0trap\t\r \u00a0mass\t\r \u00a0spectrometry.\t\r \u00a0Anal\t\r \u00a0Chem\t\r \u00a076,\t\r \u00a03590-\u00ad\u20103598\t\r \u00a0(2004).\t\r \u00a0421.\t\r \u00a0 H.\t\r \u00a0Steen,\t\r \u00a0B.\t\r \u00a0Kuster,\t\r \u00a0M.\t\r \u00a0Fernandez,\t\r \u00a0A.\t\r \u00a0Pandey,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Tyrosine\t\r \u00a0phosphorylation\t\r \u00a0mapping\t\r \u00a0of\t\r \u00a0the\t\r \u00a0epidermal\t\r \u00a0growth\t\r \u00a0factor\t\r \u00a0receptor\t\r \u00a0signaling\t\r \u00a0pathway.\t\r \u00a0J\t\r \u00a0Biol\t\r \u00a0Chem\t\r \u00a0277,\t\r \u00a01031-\u00ad\u20101039\t\r \u00a0(2002).\t\r \u00a0422.\t\r \u00a0 J.\t\r \u00a0Villen,\t\r \u00a0S.\t\r \u00a0A.\t\r \u00a0Beausoleil,\t\r \u00a0S.\t\r \u00a0P.\t\r \u00a0Gygi,\t\r \u00a0Evaluation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0utility\t\r \u00a0of\t\r \u00a0neutral-\u00ad\u2010loss-\u00ad\u2010dependent\t\r \u00a0MS3\t\r \u00a0strategies\t\r \u00a0in\t\r \u00a0large-\u00ad\u2010scale\t\r \u00a0phosphorylation\t\r \u00a0analysis.\t\r \u00a0 Proteomics\t\r \u00a08,\t\r \u00a04444-\u00ad\u20104452\t\r \u00a0(2008).\t\r \u00a0423.\t\r \u00a0 P.\t\r \u00a0J.\t\r \u00a0Ulintz,\t\r \u00a0A.\t\r \u00a0K.\t\r \u00a0Yocum,\t\r \u00a0B.\t\r \u00a0Bodenmiller,\t\r \u00a0R.\t\r \u00a0Aebersold,\t\r \u00a0P.\t\r \u00a0C.\t\r \u00a0Andrews,\t\r \u00a0A.\t\r \u00a0I.\t\r \u00a0Nesvizhskii,\t\r \u00a0Comparison\t\r \u00a0of\t\r \u00a0MS(2)-\u00ad\u2010Only,\t\r \u00a0MSA,\t\r \u00a0and\t\r \u00a0MS(2)\/MS(3)\t\r \u00a0 \t\r \u00a0\t\r \u00a0 175\t\r \u00a0 Methodologies\t\r \u00a0for\t\r \u00a0Phosphopeptide\t\r \u00a0Identification.\t\r \u00a0J\t\r \u00a0Proteome\t\r \u00a0Res\t\r \u00a08,\t\r \u00a0887-\u00ad\u2010899\t\r \u00a0(2009).\t\r \u00a0424.\t\r \u00a0 R.\t\r \u00a0Kr\u00fcger,\t\r \u00a0C.-\u00ad\u2010W.\t\r \u00a0Hung,\t\r \u00a0M.\t\r \u00a0Edelson-\u00ad\u2010Averbukh,\t\r \u00a0W.\t\r \u00a0D.\t\r \u00a0Lehmann,\t\r \u00a0Iodoacetamide-\u00ad\u2010alkylated\t\r \u00a0methionine\t\r \u00a0can\t\r \u00a0mimic\t\r \u00a0neutral\t\r \u00a0loss\t\r \u00a0of\t\r \u00a0phosphoric\t\r \u00a0acid\t\r \u00a0from\t\r \u00a0phosphopeptides\t\r \u00a0as\t\r \u00a0exemplified\t\r \u00a0by\t\r \u00a0nano-\u00ad\u2010electrospray\t\r \u00a0ionization\t\r \u00a0quadrupole\t\r \u00a0time-\u00ad\u2010of-\u00ad\u2010flight\t\r \u00a0parent\t\r \u00a0ion\t\r \u00a0scanning.\t\r \u00a0Rapid\t\r \u00a0Commun.\t\r \u00a0Mass\t\r \u00a0Spectrom.\t\r \u00a019,\t\r \u00a01709-\u00ad\u20101716\t\r \u00a0(2005).\t\r \u00a0425.\t\r \u00a0 A.\t\r \u00a0M.\t\r \u00a0Palumbo,\t\r \u00a0G.\t\r \u00a0E.\t\r \u00a0Reid,\t\r \u00a0Evaluation\t\r \u00a0of\t\r \u00a0Gas-\u00ad\u2010Phase\t\r \u00a0Rearrangement\t\r \u00a0and\t\r \u00a0Competing\t\r \u00a0Fragmentation\t\r \u00a0Reactions\t\r \u00a0on\t\r \u00a0Protein\t\r \u00a0Phosphorylation\t\r \u00a0Site\t\r \u00a0Assignment\t\r \u00a0Using\t\r \u00a0Collision\t\r \u00a0Induced\t\r \u00a0Dissociation-\u00ad\u2010MS\/MS\t\r \u00a0and\t\r \u00a0MS(3).\t\r \u00a0Anal\t\r \u00a0 Chem,\t\r \u00a0\t\r \u00a0(2008).\t\r \u00a0426.\t\r \u00a0 H.\t\r \u00a0Molina,\t\r \u00a0D.\t\r \u00a0M.\t\r \u00a0Horn,\t\r \u00a0N.\t\r \u00a0Tang,\t\r \u00a0S.\t\r \u00a0Mathivanan,\t\r \u00a0A.\t\r \u00a0Pandey,\t\r \u00a0Global\t\r \u00a0proteomic\t\r \u00a0profiling\t\r \u00a0of\t\r \u00a0phosphopeptides\t\r \u00a0using\t\r \u00a0electron\t\r \u00a0transfer\t\r \u00a0dissociation\t\r \u00a0tandem\t\r \u00a0mass\t\r \u00a0spectrometry.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a0104,\t\r \u00a02199-\u00ad\u20102204\t\r \u00a0(2007).\t\r \u00a0427.\t\r \u00a0 A.\t\r \u00a0Stensballe,\t\r \u00a0O.\t\r \u00a0N.\t\r \u00a0Jensen,\t\r \u00a0J.\t\r \u00a0V.\t\r \u00a0Olsen,\t\r \u00a0K.\t\r \u00a0F.\t\r \u00a0Haselmann,\t\r \u00a0R.\t\r \u00a0A.\t\r \u00a0Zubarev,\t\r \u00a0Electron\t\r \u00a0capture\t\r \u00a0dissociation\t\r \u00a0of\t\r \u00a0singly\t\r \u00a0and\t\r \u00a0multiply\t\r \u00a0phosphorylated\t\r \u00a0peptides.\t\r \u00a0Rapid\t\r \u00a0 Commun\t\r \u00a0Mass\t\r \u00a0Spectrom\t\r \u00a014,\t\r \u00a01793-\u00ad\u20101800\t\r \u00a0(2000).\t\r \u00a0428.\t\r \u00a0 U.\t\r \u00a0Team,\t\r \u00a0The\t\r \u00a0Universal\t\r \u00a0Protein\t\r \u00a0Resource\t\r \u00a0(UniProt)\t\r \u00a02009.\t\r \u00a0Nucleic\t\r \u00a0Acids\t\r \u00a0Res\t\r \u00a0 37,\t\r \u00a0D169-\u00ad\u2010174\t\r \u00a0(2009).\t\r \u00a0429.\t\r \u00a0 F.\t\r \u00a0Gnad,\t\r \u00a0S.\t\r \u00a0Ren,\t\r \u00a0J.\t\r \u00a0Cox,\t\r \u00a0J.\t\r \u00a0V.\t\r \u00a0Olsen,\t\r \u00a0B.\t\r \u00a0Macek,\t\r \u00a0M.\t\r \u00a0Oroshi,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0PHOSIDA\t\r \u00a0(phosphorylation\t\r \u00a0site\t\r \u00a0database):\t\r \u00a0management,\t\r \u00a0structural\t\r \u00a0and\t\r \u00a0evolutionary\t\r \u00a0investigation,\t\r \u00a0and\t\r \u00a0prediction\t\r \u00a0of\t\r \u00a0phosphosites.\t\r \u00a0Genome\t\r \u00a0Biol\t\r \u00a08,\t\r \u00a0R250\t\r \u00a0(2007).\t\r \u00a0430.\t\r \u00a0 P.\t\r \u00a0Adams,\t\r \u00a0R.\t\r \u00a0Fowler,\t\r \u00a0N.\t\r \u00a0Kinsella,\t\r \u00a0G.\t\r \u00a0Howell,\t\r \u00a0M.\t\r \u00a0Farris,\t\r \u00a0P.\t\r \u00a0Coote,\t\r \u00a0C.\t\r \u00a0D.\t\r \u00a0O'Connor,\t\r \u00a0Proteomic\t\r \u00a0detection\t\r \u00a0of\t\r \u00a0PhoPQ-\u00ad\u2010\t\r \u00a0and\t\r \u00a0acid-\u00ad\u2010mediated\t\r \u00a0repression\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0motility.\t\r \u00a0Proteomics\t\r \u00a01,\t\r \u00a0597-\u00ad\u2010607\t\r \u00a0(2001).\t\r \u00a0431.\t\r \u00a0 D.\t\r \u00a0Agudo,\t\r \u00a0M.\t\r \u00a0T.\t\r \u00a0Mendoza,\t\r \u00a0C.\t\r \u00a0Castanares,\t\r \u00a0C.\t\r \u00a0Nombela,\t\r \u00a0R.\t\r \u00a0Rotger,\t\r \u00a0A\t\r \u00a0proteomic\t\r \u00a0approach\t\r \u00a0to\t\r \u00a0study\t\r \u00a0Salmonella\t\r \u00a0typhi\t\r \u00a0periplasmic\t\r \u00a0proteins\t\r \u00a0altered\t\r \u00a0by\t\r \u00a0a\t\r \u00a0lack\t\r \u00a0of\t\r \u00a0the\t\r \u00a0DsbA\t\r \u00a0thiol:\t\r \u00a0disulfide\t\r \u00a0isomerase.\t\r \u00a0Proteomics\t\r \u00a04,\t\r \u00a0355-\u00ad\u2010363\t\r \u00a0(2004).\t\r \u00a0432.\t\r \u00a0 P.\t\r \u00a0A.\t\r \u00a0Berk,\t\r \u00a0R.\t\r \u00a0Jonge,\t\r \u00a0M.\t\r \u00a0H.\t\r \u00a0Zwietering,\t\r \u00a0T.\t\r \u00a0Abee,\t\r \u00a0J.\t\r \u00a0Kieboom,\t\r \u00a0Acid\t\r \u00a0resistance\t\r \u00a0variability\t\r \u00a0among\t\r \u00a0isolates\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0Typhimurium\t\r \u00a0DT104.\t\r \u00a0J\t\r \u00a0Appl\t\r \u00a0Microbiol\t\r \u00a099,\t\r \u00a0859-\u00ad\u2010866\t\r \u00a0(2005).\t\r \u00a0433.\t\r \u00a0 V.\t\r \u00a0Encheva,\t\r \u00a0R.\t\r \u00a0Wait,\t\r \u00a0S.\t\r \u00a0Begum,\t\r \u00a0S.\t\r \u00a0E.\t\r \u00a0Gharbia,\t\r \u00a0H.\t\r \u00a0N.\t\r \u00a0Shah,\t\r \u00a0Protein\t\r \u00a0expression\t\r \u00a0diversity\t\r \u00a0amongst\t\r \u00a0serovars\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0enterica.\t\r \u00a0Microbiology\t\r \u00a0153,\t\r \u00a04183-\u00ad\u20104193\t\r \u00a0(2007).\t\r \u00a0434.\t\r \u00a0 K.\t\r \u00a0M.\t\r \u00a0Osman,\t\r \u00a0M.\t\r \u00a0M.\t\r \u00a0Ali,\t\r \u00a0M.\t\r \u00a0I.\t\r \u00a0Radwan,\t\r \u00a0H.\t\r \u00a0K.\t\r \u00a0Kim,\t\r \u00a0J.\t\r \u00a0Han,\t\r \u00a0Comparative\t\r \u00a0proteomic\t\r \u00a0analysis\t\r \u00a0on\t\r \u00a0Salmonella\t\r \u00a0Gallinarum\t\r \u00a0and\t\r \u00a0Salmonella\t\r \u00a0Enteritidis\t\r \u00a0exploring\t\r \u00a0proteins\t\r \u00a0that\t\r \u00a0may\t\r \u00a0incorporate\t\r \u00a0host\t\r \u00a0adaptation\t\r \u00a0in\t\r \u00a0poultry.\t\r \u00a0J\t\r \u00a0Proteomics\t\r \u00a072,\t\r \u00a0815-\u00ad\u2010821\t\r \u00a0(2009).\t\r \u00a0435.\t\r \u00a0 K.\t\r \u00a0A.\t\r \u00a0Sonck,\t\r \u00a0G.\t\r \u00a0Kint,\t\r \u00a0G.\t\r \u00a0Schoofs,\t\r \u00a0C.\t\r \u00a0Vander\t\r \u00a0Wauven,\t\r \u00a0J.\t\r \u00a0Vanderleyden,\t\r \u00a0S.\t\r \u00a0C.\t\r \u00a0De\t\r \u00a0Keersmaecker,\t\r \u00a0The\t\r \u00a0proteome\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0Typhimurium\t\r \u00a0grown\t\r \u00a0under\t\r \u00a0in\t\r \u00a0vivo-\u00ad\u2010mimicking\t\r \u00a0conditions.\t\r \u00a0Proteomics\t\r \u00a09,\t\r \u00a0565-\u00ad\u2010579\t\r \u00a0(2009).\t\r \u00a0436.\t\r \u00a0 G.\t\r \u00a0Kint,\t\r \u00a0K.\t\r \u00a0A.\t\r \u00a0Sonck,\t\r \u00a0G.\t\r \u00a0Schoofs,\t\r \u00a0D.\t\r \u00a0De\t\r \u00a0Coster,\t\r \u00a0J.\t\r \u00a0Vanderleyden,\t\r \u00a0S.\t\r \u00a0C.\t\r \u00a0De\t\r \u00a0Keersmaecker,\t\r \u00a02D\t\r \u00a0proteome\t\r \u00a0analysis\t\r \u00a0initiates\t\r \u00a0new\t\r \u00a0insights\t\r \u00a0on\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0Typhimurium\t\r \u00a0LuxS\t\r \u00a0protein.\t\r \u00a0BMC\t\r \u00a0Microbiol\t\r \u00a09,\t\r \u00a0198\t\r \u00a0(2009).\t\r \u00a0437.\t\r \u00a0 L.\t\r \u00a0Pinto,\t\r \u00a0P.\t\r \u00a0Poeta,\t\r \u00a0S.\t\r \u00a0Vieira,\t\r \u00a0C.\t\r \u00a0Caleja,\t\r \u00a0H.\t\r \u00a0Radhouani,\t\r \u00a0C.\t\r \u00a0Carvalho,\t\r \u00a0M.\t\r \u00a0Vieira-\u00ad\u2010Pinto,\t\r \u00a0P.\t\r \u00a0Themudo,\t\r \u00a0C.\t\r \u00a0Torres,\t\r \u00a0R.\t\r \u00a0Vitorino,\t\r \u00a0P.\t\r \u00a0Domingues,\t\r \u00a0G.\t\r \u00a0Igrejas,\t\r \u00a0Genomic\t\r \u00a0 \t\r \u00a0\t\r \u00a0 176\t\r \u00a0 and\t\r \u00a0proteomic\t\r \u00a0evaluation\t\r \u00a0of\t\r \u00a0antibiotic\t\r \u00a0resistance\t\r \u00a0in\t\r \u00a0Salmonella\t\r \u00a0strains.\t\r \u00a0J\t\r \u00a0 Proteomics\t\r \u00a073,\t\r \u00a01535-\u00ad\u20101541\t\r \u00a0(2010).\t\r \u00a0438.\t\r \u00a0 R.\t\r \u00a0Di\t\r \u00a0Pasqua,\t\r \u00a0G.\t\r \u00a0Mamone,\t\r \u00a0P.\t\r \u00a0Ferranti,\t\r \u00a0D.\t\r \u00a0Ercolini,\t\r \u00a0G.\t\r \u00a0Mauriello,\t\r \u00a0Changes\t\r \u00a0in\t\r \u00a0the\t\r \u00a0proteome\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0Thompson\t\r \u00a0as\t\r \u00a0stress\t\r \u00a0adaptation\t\r \u00a0to\t\r \u00a0sublethal\t\r \u00a0concentrations\t\r \u00a0of\t\r \u00a0thymol.\t\r \u00a0Proteomics\t\r \u00a010,\t\r \u00a01040-\u00ad\u20101049\t\r \u00a0(2010).\t\r \u00a0439.\t\r \u00a0 K.\t\r \u00a0Kim,\t\r \u00a0E.\t\r \u00a0Yang,\t\r \u00a0G.\t\r \u00a0P.\t\r \u00a0Vu,\t\r \u00a0H.\t\r \u00a0Gong,\t\r \u00a0J.\t\r \u00a0Su,\t\r \u00a0F.\t\r \u00a0Liu,\t\r \u00a0S.\t\r \u00a0Lu,\t\r \u00a0Mass\t\r \u00a0spectrometry-\u00ad\u2010based\t\r \u00a0quantitative\t\r \u00a0proteomic\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0Enteritidis\t\r \u00a0protein\t\r \u00a0expression\t\r \u00a0upon\t\r \u00a0exposure\t\r \u00a0to\t\r \u00a0hydrogen\t\r \u00a0peroxide.\t\r \u00a0BMC\t\r \u00a0Microbiol\t\r \u00a010,\t\r \u00a0166\t\r \u00a0(2010).\t\r \u00a0440.\t\r \u00a0 C.\t\r \u00a0Ansong,\t\r \u00a0H.\t\r \u00a0Yoon,\t\r \u00a0S.\t\r \u00a0Porwollik,\t\r \u00a0H.\t\r \u00a0Mottaz-\u00ad\u2010Brewer,\t\r \u00a0B.\t\r \u00a0O.\t\r \u00a0Petritis,\t\r \u00a0N.\t\r \u00a0Jaitly,\t\r \u00a0J.\t\r \u00a0N.\t\r \u00a0Adkins,\t\r \u00a0M.\t\r \u00a0McClelland,\t\r \u00a0F.\t\r \u00a0Heffron,\t\r \u00a0R.\t\r \u00a0D.\t\r \u00a0Smith,\t\r \u00a0Global\t\r \u00a0systems-\u00ad\u2010level\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0Hfq\t\r \u00a0and\t\r \u00a0SmpB\t\r \u00a0deletion\t\r \u00a0mutants\t\r \u00a0in\t\r \u00a0Salmonella:\t\r \u00a0implications\t\r \u00a0for\t\r \u00a0virulence\t\r \u00a0and\t\r \u00a0global\t\r \u00a0protein\t\r \u00a0translation.\t\r \u00a0PLoS\t\r \u00a0One\t\r \u00a04,\t\r \u00a0e4809\t\r \u00a0(2009).\t\r \u00a0441.\t\r \u00a0 R.\t\r \u00a0C.\t\r \u00a0Charles,\t\r \u00a0J.\t\r \u00a0B.\t\r \u00a0Harris,\t\r \u00a0M.\t\r \u00a0R.\t\r \u00a0Chase,\t\r \u00a0L.\t\r \u00a0M.\t\r \u00a0Lebrun,\t\r \u00a0A.\t\r \u00a0Sheikh,\t\r \u00a0R.\t\r \u00a0C.\t\r \u00a0LaRocque,\t\r \u00a0T.\t\r \u00a0Logvinenko,\t\r \u00a0S.\t\r \u00a0M.\t\r \u00a0Rollins,\t\r \u00a0A.\t\r \u00a0Tarique,\t\r \u00a0E.\t\r \u00a0L.\t\r \u00a0Hohmann,\t\r \u00a0I.\t\r \u00a0Rosenberg,\t\r \u00a0B.\t\r \u00a0Krastins,\t\r \u00a0D.\t\r \u00a0A.\t\r \u00a0Sarracino,\t\r \u00a0F.\t\r \u00a0Qadri,\t\r \u00a0S.\t\r \u00a0B.\t\r \u00a0Calderwood,\t\r \u00a0E.\t\r \u00a0T.\t\r \u00a0Ryan,\t\r \u00a0Comparative\t\r \u00a0proteomic\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0the\t\r \u00a0PhoP\t\r \u00a0regulon\t\r \u00a0in\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0Typhi\t\r \u00a0versus\t\r \u00a0Typhimurium.\t\r \u00a0PLoS\t\r \u00a0One\t\r \u00a04,\t\r \u00a0e6994\t\r \u00a0(2009).\t\r \u00a0442.\t\r \u00a0 D.\t\r \u00a0Ciavardelli,\t\r \u00a0S.\t\r \u00a0Ammendola,\t\r \u00a0M.\t\r \u00a0Ronci,\t\r \u00a0A.\t\r \u00a0Consalvo,\t\r \u00a0V.\t\r \u00a0Marzano,\t\r \u00a0M.\t\r \u00a0Lipoma,\t\r \u00a0P.\t\r \u00a0Sacchetta,\t\r \u00a0G.\t\r \u00a0Federici,\t\r \u00a0C.\t\r \u00a0Di\t\r \u00a0Ilio,\t\r \u00a0A.\t\r \u00a0Battistoni,\t\r \u00a0A.\t\r \u00a0Urbani,\t\r \u00a0Phenotypic\t\r \u00a0profile\t\r \u00a0linked\t\r \u00a0to\t\r \u00a0inhibition\t\r \u00a0of\t\r \u00a0the\t\r \u00a0major\t\r \u00a0Zn\t\r \u00a0influx\t\r \u00a0system\t\r \u00a0in\t\r \u00a0Salmonella\t\r \u00a0enterica:\t\r \u00a0proteomics\t\r \u00a0and\t\r \u00a0ionomics\t\r \u00a0investigations.\t\r \u00a0Mol\t\r \u00a0Biosyst,\t\r \u00a0\t\r \u00a0(2010).\t\r \u00a0443.\t\r \u00a0 R.\t\r \u00a0C.\t\r \u00a0Charles,\t\r \u00a0A.\t\r \u00a0Sheikh,\t\r \u00a0B.\t\r \u00a0Krastins,\t\r \u00a0J.\t\r \u00a0B.\t\r \u00a0Harris,\t\r \u00a0M.\t\r \u00a0S.\t\r \u00a0Bhuiyan,\t\r \u00a0R.\t\r \u00a0C.\t\r \u00a0LaRocque,\t\r \u00a0T.\t\r \u00a0Logvinenko,\t\r \u00a0D.\t\r \u00a0A.\t\r \u00a0Sarracino,\t\r \u00a0I.\t\r \u00a0T.\t\r \u00a0Kudva,\t\r \u00a0J.\t\r \u00a0Eisenstein,\t\r \u00a0M.\t\r \u00a0J.\t\r \u00a0Podolsky,\t\r \u00a0A.\t\r \u00a0Kalsy,\t\r \u00a0W.\t\r \u00a0A.\t\r \u00a0Brooks,\t\r \u00a0A.\t\r \u00a0Ludwig,\t\r \u00a0M.\t\r \u00a0John,\t\r \u00a0S.\t\r \u00a0B.\t\r \u00a0Calderwood,\t\r \u00a0F.\t\r \u00a0Qadri,\t\r \u00a0E.\t\r \u00a0T.\t\r \u00a0Ryan,\t\r \u00a0Characterization\t\r \u00a0of\t\r \u00a0anti-\u00ad\u2010Salmonella\t\r \u00a0enterica\t\r \u00a0serotype\t\r \u00a0Typhi\t\r \u00a0antibody\t\r \u00a0responses\t\r \u00a0in\t\r \u00a0bacteremic\t\r \u00a0Bangladeshi\t\r \u00a0patients\t\r \u00a0by\t\r \u00a0an\t\r \u00a0immunoaffinity\t\r \u00a0proteomics-\u00ad\u2010based\t\r \u00a0technology.\t\r \u00a0Clin\t\r \u00a0Vaccine\t\r \u00a0Immunol\t\r \u00a017,\t\r \u00a01188-\u00ad\u20101195\t\r \u00a0(2010).\t\r \u00a0444.\t\r \u00a0 D.\t\r \u00a0Becker,\t\r \u00a0M.\t\r \u00a0Selbach,\t\r \u00a0C.\t\r \u00a0Rollenhagen,\t\r \u00a0M.\t\r \u00a0Ballmaier,\t\r \u00a0T.\t\r \u00a0F.\t\r \u00a0Meyer,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0D.\t\r \u00a0Bumann,\t\r \u00a0Robust\t\r \u00a0Salmonella\t\r \u00a0metabolism\t\r \u00a0limits\t\r \u00a0possibilities\t\r \u00a0for\t\r \u00a0new\t\r \u00a0antimicrobials.\t\r \u00a0Nature\t\r \u00a0440,\t\r \u00a0303-\u00ad\u2010307\t\r \u00a0(2006).\t\r \u00a0445.\t\r \u00a0 L.\t\r \u00a0Shi,\t\r \u00a0J.\t\r \u00a0N.\t\r \u00a0Adkins,\t\r \u00a0J.\t\r \u00a0R.\t\r \u00a0Coleman,\t\r \u00a0A.\t\r \u00a0A.\t\r \u00a0Schepmoes,\t\r \u00a0A.\t\r \u00a0Dohnkova,\t\r \u00a0H.\t\r \u00a0M.\t\r \u00a0Mottaz,\t\r \u00a0A.\t\r \u00a0D.\t\r \u00a0Norbeck,\t\r \u00a0S.\t\r \u00a0O.\t\r \u00a0Purvine,\t\r \u00a0N.\t\r \u00a0P.\t\r \u00a0Manes,\t\r \u00a0H.\t\r \u00a0S.\t\r \u00a0Smallwood,\t\r \u00a0H.\t\r \u00a0Wang,\t\r \u00a0J.\t\r \u00a0Forbes,\t\r \u00a0P.\t\r \u00a0Gros,\t\r \u00a0S.\t\r \u00a0Uzzau,\t\r \u00a0K.\t\r \u00a0D.\t\r \u00a0Rodland,\t\r \u00a0F.\t\r \u00a0Heffron,\t\r \u00a0R.\t\r \u00a0D.\t\r \u00a0Smith,\t\r \u00a0T.\t\r \u00a0C.\t\r \u00a0Squier,\t\r \u00a0Proteomic\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0typhimurium\t\r \u00a0isolated\t\r \u00a0from\t\r \u00a0RAW\t\r \u00a0264.7\t\r \u00a0macrophages:\t\r \u00a0identification\t\r \u00a0of\t\r \u00a0a\t\r \u00a0novel\t\r \u00a0protein\t\r \u00a0that\t\r \u00a0contributes\t\r \u00a0to\t\r \u00a0the\t\r \u00a0replication\t\r \u00a0of\t\r \u00a0serovar\t\r \u00a0typhimurium\t\r \u00a0inside\t\r \u00a0macrophages.\t\r \u00a0J\t\r \u00a0Biol\t\r \u00a0Chem\t\r \u00a0281,\t\r \u00a029131-\u00ad\u201029140\t\r \u00a0(2006).\t\r \u00a0446.\t\r \u00a0 L.\t\r \u00a0Shi,\t\r \u00a0S.\t\r \u00a0M.\t\r \u00a0Chowdhury,\t\r \u00a0H.\t\r \u00a0S.\t\r \u00a0Smallwood,\t\r \u00a0H.\t\r \u00a0Yoon,\t\r \u00a0H.\t\r \u00a0M.\t\r \u00a0Mottaz-\u00ad\u2010Brewer,\t\r \u00a0A.\t\r \u00a0D.\t\r \u00a0Norbeck,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0McDermott,\t\r \u00a0T.\t\r \u00a0R.\t\r \u00a0Clauss,\t\r \u00a0F.\t\r \u00a0Heffron,\t\r \u00a0R.\t\r \u00a0D.\t\r \u00a0Smith,\t\r \u00a0J.\t\r \u00a0N.\t\r \u00a0Adkins,\t\r \u00a0Proteomic\t\r \u00a0investigation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0time\t\r \u00a0course\t\r \u00a0responses\t\r \u00a0of\t\r \u00a0RAW\t\r \u00a0264.7\t\r \u00a0macrophages\t\r \u00a0to\t\r \u00a0infection\t\r \u00a0with\t\r \u00a0Salmonella\t\r \u00a0enterica.\t\r \u00a0Infect\t\r \u00a0Immun\t\r \u00a077,\t\r \u00a03227-\u00ad\u20103233\t\r \u00a0(2009).\t\r \u00a0447.\t\r \u00a0 C.\t\r \u00a0Molero,\t\r \u00a0I.\t\r \u00a0Rodriguez-\u00ad\u2010Escudero,\t\r \u00a0A.\t\r \u00a0Aleman,\t\r \u00a0R.\t\r \u00a0Rotger,\t\r \u00a0M.\t\r \u00a0Molina,\t\r \u00a0V.\t\r \u00a0J.\t\r \u00a0Cid,\t\r \u00a0Addressing\t\r \u00a0the\t\r \u00a0effects\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0internalization\t\r \u00a0in\t\r \u00a0host\t\r \u00a0cell\t\r \u00a0signaling\t\r \u00a0on\t\r \u00a0a\t\r \u00a0reverse-\u00ad\u2010phase\t\r \u00a0protein\t\r \u00a0array.\t\r \u00a0Proteomics\t\r \u00a09,\t\r \u00a03652-\u00ad\u20103665\t\r \u00a0(2009).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 177\t\r \u00a0 448.\t\r \u00a0 J.\t\r \u00a0N.\t\r \u00a0Adkins,\t\r \u00a0H.\t\r \u00a0M.\t\r \u00a0Mottaz,\t\r \u00a0A.\t\r \u00a0D.\t\r \u00a0Norbeck,\t\r \u00a0J.\t\r \u00a0K.\t\r \u00a0Gustin,\t\r \u00a0J.\t\r \u00a0Rue,\t\r \u00a0T.\t\r \u00a0R.\t\r \u00a0Clauss,\t\r \u00a0S.\t\r \u00a0O.\t\r \u00a0Purvine,\t\r \u00a0K.\t\r \u00a0D.\t\r \u00a0Rodland,\t\r \u00a0F.\t\r \u00a0Heffron,\t\r \u00a0R.\t\r \u00a0D.\t\r \u00a0Smith,\t\r \u00a0Analysis\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0proteome\t\r \u00a0through\t\r \u00a0environmental\t\r \u00a0response\t\r \u00a0toward\t\r \u00a0infectious\t\r \u00a0conditions.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Proteomics\t\r \u00a05,\t\r \u00a01450-\u00ad\u20101461\t\r \u00a0(2006).\t\r \u00a0449.\t\r \u00a0 N.\t\r \u00a0P.\t\r \u00a0Manes,\t\r \u00a0J.\t\r \u00a0K.\t\r \u00a0Gustin,\t\r \u00a0J.\t\r \u00a0Rue,\t\r \u00a0H.\t\r \u00a0M.\t\r \u00a0Mottaz,\t\r \u00a0S.\t\r \u00a0O.\t\r \u00a0Purvine,\t\r \u00a0A.\t\r \u00a0D.\t\r \u00a0Norbeck,\t\r \u00a0M.\t\r \u00a0E.\t\r \u00a0Monroe,\t\r \u00a0J.\t\r \u00a0S.\t\r \u00a0Zimmer,\t\r \u00a0T.\t\r \u00a0O.\t\r \u00a0Metz,\t\r \u00a0J.\t\r \u00a0N.\t\r \u00a0Adkins,\t\r \u00a0R.\t\r \u00a0D.\t\r \u00a0Smith,\t\r \u00a0F.\t\r \u00a0Heffron,\t\r \u00a0Targeted\t\r \u00a0protein\t\r \u00a0degradation\t\r \u00a0by\t\r \u00a0Salmonella\t\r \u00a0under\t\r \u00a0phagosome-\u00ad\u2010mimicking\t\r \u00a0culture\t\r \u00a0conditions\t\r \u00a0investigated\t\r \u00a0using\t\r \u00a0comparative\t\r \u00a0peptidomics.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Proteomics\t\r \u00a0 6,\t\r \u00a0717-\u00ad\u2010727\t\r \u00a0(2007).\t\r \u00a0450.\t\r \u00a0 C.\t\r \u00a0Ansong,\t\r \u00a0H.\t\r \u00a0Yoon,\t\r \u00a0A.\t\r \u00a0D.\t\r \u00a0Norbeck,\t\r \u00a0J.\t\r \u00a0K.\t\r \u00a0Gustin,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0McDermott,\t\r \u00a0H.\t\r \u00a0M.\t\r \u00a0Mottaz,\t\r \u00a0J.\t\r \u00a0Rue,\t\r \u00a0J.\t\r \u00a0N.\t\r \u00a0Adkins,\t\r \u00a0F.\t\r \u00a0Heffron,\t\r \u00a0R.\t\r \u00a0D.\t\r \u00a0Smith,\t\r \u00a0Proteomics\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0the\t\r \u00a0causative\t\r \u00a0agent\t\r \u00a0of\t\r \u00a0typhoid\t\r \u00a0fever.\t\r \u00a0J\t\r \u00a0Proteome\t\r \u00a0Res\t\r \u00a07,\t\r \u00a0546-\u00ad\u2010557\t\r \u00a0(2008).\t\r \u00a0451.\t\r \u00a0 N.\t\r \u00a0S.\t\r \u00a0Galbraith,\t\r \u00a0Studies\t\r \u00a0of\t\r \u00a0human\t\r \u00a0Salmonellosis\t\r \u00a0in\t\r \u00a0relation\t\r \u00a0to\t\r \u00a0infection\t\r \u00a0in\t\r \u00a0animals.\t\r \u00a0Vet\t\r \u00a0Rec\t\r \u00a073,\t\r \u00a01296-\u00ad\u20101303\t\r \u00a0(1961).\t\r \u00a0452.\t\r \u00a0 R.\t\r \u00a0L.\t\r \u00a0Santos,\t\r \u00a0S.\t\r \u00a0Zhang,\t\r \u00a0R.\t\r \u00a0M.\t\r \u00a0Tsolis,\t\r \u00a0R.\t\r \u00a0A.\t\r \u00a0Kingsley,\t\r \u00a0L.\t\r \u00a0G.\t\r \u00a0Adams,\t\r \u00a0A.\t\r \u00a0J.\t\r \u00a0Baumler,\t\r \u00a0Animal\t\r \u00a0models\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0infections:\t\r \u00a0enteritis\t\r \u00a0versus\t\r \u00a0typhoid\t\r \u00a0fever.\t\r \u00a0 Microbes\t\r \u00a0Infect\t\r \u00a03,\t\r \u00a01335-\u00ad\u20101344\t\r \u00a0(2001).\t\r \u00a0453.\t\r \u00a0 S.\t\r \u00a0Hashim,\t\r \u00a0K.\t\r \u00a0Mukherjee,\t\r \u00a0M.\t\r \u00a0Raje,\t\r \u00a0S.\t\r \u00a0K.\t\r \u00a0Basu,\t\r \u00a0A.\t\r \u00a0Mukhopadhyay,\t\r \u00a0Live\t\r \u00a0Salmonella\t\r \u00a0modulate\t\r \u00a0expression\t\r \u00a0of\t\r \u00a0Rab\t\r \u00a0proteins\t\r \u00a0to\t\r \u00a0persist\t\r \u00a0in\t\r \u00a0a\t\r \u00a0specialized\t\r \u00a0compartment\t\r \u00a0and\t\r \u00a0escape\t\r \u00a0transport\t\r \u00a0to\t\r \u00a0lysosomes.\t\r \u00a0J\t\r \u00a0Biol\t\r \u00a0Chem\t\r \u00a0275,\t\r \u00a016281-\u00ad\u201016288\t\r \u00a0(2000).\t\r \u00a0454.\t\r \u00a0 L.\t\r \u00a0A.\t\r \u00a0Knodler,\t\r \u00a0O.\t\r \u00a0Steele-\u00ad\u2010Mortimer,\t\r \u00a0Taking\t\r \u00a0possession:\t\r \u00a0biogenesis\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Salmonella-\u00ad\u2010containing\t\r \u00a0vacuole.\t\r \u00a0Traffic\t\r \u00a04,\t\r \u00a0587-\u00ad\u2010599\t\r \u00a0(2003).\t\r \u00a0455.\t\r \u00a0 S.\t\r \u00a0R.\t\r \u00a0Waterman,\t\r \u00a0D.\t\r \u00a0W.\t\r \u00a0Holden,\t\r \u00a0Functions\t\r \u00a0and\t\r \u00a0effectors\t\r \u00a0of\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0pathogenicity\t\r \u00a0island\t\r \u00a02\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion\t\r \u00a0system.\t\r \u00a0Cell\t\r \u00a0Microbiol\t\r \u00a05,\t\r \u00a0501-\u00ad\u2010511\t\r \u00a0(2003).\t\r \u00a0456.\t\r \u00a0 D.\t\r \u00a0G.\t\r \u00a0Guiney,\t\r \u00a0M.\t\r \u00a0Lesnick,\t\r \u00a0Targeting\t\r \u00a0of\t\r \u00a0the\t\r \u00a0actin\t\r \u00a0cytoskeleton\t\r \u00a0during\t\r \u00a0infection\t\r \u00a0by\t\r \u00a0Salmonella\t\r \u00a0strains.\t\r \u00a0Clin\t\r \u00a0Immunol\t\r \u00a0114,\t\r \u00a0248-\u00ad\u2010255\t\r \u00a0(2005).\t\r \u00a0457.\t\r \u00a0 J.\t\r \u00a0C.\t\r \u00a0Patel,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Manipulation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0host\t\r \u00a0actin\t\r \u00a0cytoskeleton\t\r \u00a0by\t\r \u00a0Salmonella-\u00ad\u2010-\u00ad\u2010all\t\r \u00a0in\t\r \u00a0the\t\r \u00a0name\t\r \u00a0of\t\r \u00a0entry.\t\r \u00a0Curr\t\r \u00a0Opin\t\r \u00a0Microbiol\t\r \u00a08,\t\r \u00a010-\u00ad\u201015\t\r \u00a0(2005).\t\r \u00a0458.\t\r \u00a0 S.\t\r \u00a0L.\t\r \u00a0Marcus,\t\r \u00a0L.\t\r \u00a0A.\t\r \u00a0Knodler,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0Salmonella\t\r \u00a0enterica\t\r \u00a0serovar\t\r \u00a0Typhimurium\t\r \u00a0effector\t\r \u00a0SigD\/SopB\t\r \u00a0is\t\r \u00a0membrane-\u00ad\u2010associated\t\r \u00a0and\t\r \u00a0ubiquitinated\t\r \u00a0inside\t\r \u00a0host\t\r \u00a0cells.\t\r \u00a0Cell\t\r \u00a0Microbiol\t\r \u00a04,\t\r \u00a0435-\u00ad\u2010446\t\r \u00a0(2002).\t\r \u00a0459.\t\r \u00a0 A.\t\r \u00a0Aleman,\t\r \u00a0I.\t\r \u00a0Rodriguez-\u00ad\u2010Escudero,\t\r \u00a0G.\t\r \u00a0V.\t\r \u00a0Mallo,\t\r \u00a0V.\t\r \u00a0J.\t\r \u00a0Cid,\t\r \u00a0M.\t\r \u00a0Molina,\t\r \u00a0R.\t\r \u00a0Rotger,\t\r \u00a0The\t\r \u00a0amino-\u00ad\u2010terminal\t\r \u00a0non-\u00ad\u2010catalytic\t\r \u00a0region\t\r \u00a0of\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0SigD\t\r \u00a0affects\t\r \u00a0actin\t\r \u00a0organization\t\r \u00a0in\t\r \u00a0yeast\t\r \u00a0and\t\r \u00a0mammalian\t\r \u00a0cells.\t\r \u00a0Cell\t\r \u00a0Microbiol\t\r \u00a07,\t\r \u00a01432-\u00ad\u20101446\t\r \u00a0(2005).\t\r \u00a0460.\t\r \u00a0 R.\t\r \u00a0J.\t\r \u00a0Botelho,\t\r \u00a0M.\t\r \u00a0Teruel,\t\r \u00a0R.\t\r \u00a0Dierckman,\t\r \u00a0R.\t\r \u00a0Anderson,\t\r \u00a0A.\t\r \u00a0Wells,\t\r \u00a0J.\t\r \u00a0D.\t\r \u00a0York,\t\r \u00a0T.\t\r \u00a0Meyer,\t\r \u00a0S.\t\r \u00a0Grinstein,\t\r \u00a0Localized\t\r \u00a0biphasic\t\r \u00a0changes\t\r \u00a0in\t\r \u00a0phosphatidylinositol-\u00ad\u20104,5-\u00ad\u2010bisphosphate\t\r \u00a0at\t\r \u00a0sites\t\r \u00a0of\t\r \u00a0phagocytosis.\t\r \u00a0J\t\r \u00a0Cell\t\r \u00a0Biol\t\r \u00a0151,\t\r \u00a01353-\u00ad\u20101368\t\r \u00a0(2000).\t\r \u00a0461.\t\r \u00a0 O.\t\r \u00a0Steele-\u00ad\u2010Mortimer,\t\r \u00a0L.\t\r \u00a0A.\t\r \u00a0Knodler,\t\r \u00a0S.\t\r \u00a0L.\t\r \u00a0Marcus,\t\r \u00a0M.\t\r \u00a0P.\t\r \u00a0Scheid,\t\r \u00a0B.\t\r \u00a0Goh,\t\r \u00a0C.\t\r \u00a0G.\t\r \u00a0Pfeifer,\t\r \u00a0V.\t\r \u00a0Duronio,\t\r \u00a0B.\t\r \u00a0B.\t\r \u00a0Finlay,\t\r \u00a0Activation\t\r \u00a0of\t\r \u00a0Akt\/protein\t\r \u00a0kinase\t\r \u00a0B\t\r \u00a0in\t\r \u00a0epithelial\t\r \u00a0cells\t\r \u00a0by\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0typhimurium\t\r \u00a0effector\t\r \u00a0sigD.\t\r \u00a0J\t\r \u00a0Biol\t\r \u00a0Chem\t\r \u00a0275,\t\r \u00a037718-\u00ad\u201037724\t\r \u00a0(2000).\t\r \u00a0462.\t\r \u00a0 I.\t\r \u00a0Rodriguez-\u00ad\u2010Escudero,\t\r \u00a0R.\t\r \u00a0Rotger,\t\r \u00a0V.\t\r \u00a0J.\t\r \u00a0Cid,\t\r \u00a0M.\t\r \u00a0Molina,\t\r \u00a0Inhibition\t\r \u00a0of\t\r \u00a0Cdc42-\u00ad\u2010dependent\t\r \u00a0signalling\t\r \u00a0in\t\r \u00a0Saccharomyces\t\r \u00a0cerevisiae\t\r \u00a0by\t\r \u00a0phosphatase-\u00ad\u2010dead\t\r \u00a0 \t\r \u00a0\t\r \u00a0 178\t\r \u00a0 SigD\/SopB\t\r \u00a0from\t\r \u00a0Salmonella\t\r \u00a0typhimurium.\t\r \u00a0Microbiology\t\r \u00a0152,\t\r \u00a03437-\u00ad\u20103452\t\r \u00a0(2006).\t\r \u00a0463.\t\r \u00a0 L.\t\r \u00a0J.\t\r \u00a0Foster,\t\r \u00a0C.\t\r \u00a0L.\t\r \u00a0de\t\r \u00a0Hoog,\t\r \u00a0Y.\t\r \u00a0Zhang,\t\r \u00a0Y.\t\r \u00a0Zhang,\t\r \u00a0X.\t\r \u00a0Xie,\t\r \u00a0V.\t\r \u00a0K.\t\r \u00a0Mootha,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0A\t\r \u00a0mammalian\t\r \u00a0organelle\t\r \u00a0map\t\r \u00a0by\t\r \u00a0protein\t\r \u00a0correlation\t\r \u00a0profiling.\t\r \u00a0Cell\t\r \u00a0125,\t\r \u00a0187-\u00ad\u2010199\t\r \u00a0(2006).\t\r \u00a0464.\t\r \u00a0 B.\t\r \u00a0Blagoev,\t\r \u00a0I.\t\r \u00a0Kratchmarova,\t\r \u00a0S.\t\r \u00a0E.\t\r \u00a0Ong,\t\r \u00a0M.\t\r \u00a0Nielsen,\t\r \u00a0L.\t\r \u00a0J.\t\r \u00a0Foster,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0A\t\r \u00a0proteomics\t\r \u00a0strategy\t\r \u00a0to\t\r \u00a0elucidate\t\r \u00a0functional\t\r \u00a0protein-\u00ad\u2010protein\t\r \u00a0interactions\t\r \u00a0applied\t\r \u00a0to\t\r \u00a0EGF\t\r \u00a0signaling.\t\r \u00a0Nat.\t\r \u00a0Biotechnol.\t\r \u00a021,\t\r \u00a0315-\u00ad\u2010318\t\r \u00a0(2003).\t\r \u00a0465.\t\r \u00a0 W.\t\r \u00a0X.\t\r \u00a0Schulze,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0A\t\r \u00a0novel\t\r \u00a0proteomic\t\r \u00a0screen\t\r \u00a0for\t\r \u00a0peptide-\u00ad\u2010protein\t\r \u00a0interactions.\t\r \u00a0J\t\r \u00a0Biol\t\r \u00a0Chem\t\r \u00a0279,\t\r \u00a010756-\u00ad\u201010764\t\r \u00a0(2004).\t\r \u00a0466.\t\r \u00a0 J.\t\r \u00a0C.\t\r \u00a0Stone,\t\r \u00a0M.\t\r \u00a0F.\t\r \u00a0Moran,\t\r \u00a0T.\t\r \u00a0Pawson,\t\r \u00a0Construction\t\r \u00a0and\t\r \u00a0expression\t\r \u00a0of\t\r \u00a0linker\t\r \u00a0insertion\t\r \u00a0and\t\r \u00a0site-\u00ad\u2010directed\t\r \u00a0mutants\t\r \u00a0of\t\r \u00a0v-\u00ad\u2010fps\t\r \u00a0protein-\u00ad\u2010tyrosine\t\r \u00a0kinase.\t\r \u00a0Methods\t\r \u00a0 Enzymol\t\r \u00a0200,\t\r \u00a0673-\u00ad\u2010692\t\r \u00a0(1991).\t\r \u00a0467.\t\r \u00a0 L.\t\r \u00a0J.\t\r \u00a0Foster,\t\r \u00a0C.\t\r \u00a0L.\t\r \u00a0De\t\r \u00a0Hoog,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Unbiased\t\r \u00a0quantitative\t\r \u00a0proteomics\t\r \u00a0of\t\r \u00a0lipid\t\r \u00a0rafts\t\r \u00a0reveals\t\r \u00a0high\t\r \u00a0specificity\t\r \u00a0for\t\r \u00a0signaling\t\r \u00a0factors.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a0 100,\t\r \u00a05813-\u00ad\u20105818\t\r \u00a0(2003).\t\r \u00a0468.\t\r \u00a0 A.\t\r \u00a0Shevchenko,\t\r \u00a0M.\t\r \u00a0Wilm,\t\r \u00a0O.\t\r \u00a0Vorm,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Mass\t\r \u00a0spectrometric\t\r \u00a0sequencing\t\r \u00a0of\t\r \u00a0proteins\t\r \u00a0silver-\u00ad\u2010stained\t\r \u00a0polyacrylamide\t\r \u00a0gels.\t\r \u00a0Anal.\t\r \u00a0Chem.\t\r \u00a068,\t\r \u00a0850-\u00ad\u2010858\t\r \u00a0(1996).\t\r \u00a0469.\t\r \u00a0 J.\t\r \u00a0Rappsilber,\t\r \u00a0Y.\t\r \u00a0Ishihama,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Stage(STop\t\r \u00a0And\t\r \u00a0Go\t\r \u00a0Extraction)\t\r \u00a0tips\t\r \u00a0for\t\r \u00a0MALDI,\t\r \u00a0nanoelectrospray,\t\r \u00a0and\t\r \u00a0LC\/MS\t\r \u00a0sample\t\r \u00a0pre-\u00ad\u2010treatment\t\r \u00a0in\t\r \u00a0proteomics.\t\r \u00a0 Anal.\t\r \u00a0Chem.\t\r \u00a0I75,\t\r \u00a0663-\u00ad\u2010670\t\r \u00a0(2003).\t\r \u00a0470.\t\r \u00a0 P.\t\r \u00a0W.\t\r \u00a0Marks,\t\r \u00a0D.\t\r \u00a0J.\t\r \u00a0Kwiatkowski,\t\r \u00a0Genomic\t\r \u00a0organization\t\r \u00a0and\t\r \u00a0chromosomal\t\r \u00a0location\t\r \u00a0of\t\r \u00a0murine\t\r \u00a0Cdc42.\t\r \u00a0Genomics\t\r \u00a038,\t\r \u00a013-\u00ad\u201018\t\r \u00a0(1996).\t\r \u00a0471.\t\r \u00a0 H.\t\r \u00a0L.\t\r \u00a0Joseph\t\r \u00a0D.\t\r \u00a0Dukes,\t\r \u00a0*\t\r \u00a0Rachel\t\r \u00a0Hagen,*\t\r \u00a0Barbara\t\r \u00a0J.\t\r \u00a0Reaves,*\t\r \u00a0Abigail\t\r \u00a0N.\t\r \u00a0Layton,\u2020\t\r \u00a0Edouard\t\r \u00a0E.\t\r \u00a0Galyov,\u2020\t\r \u00a0and\t\r \u00a0Paul\t\r \u00a0Whitley*1,\t\r \u00a0The\t\r \u00a0secreted\t\r \u00a0Salmonella\t\r \u00a0dublin\t\r \u00a0phosphoinositide\t\r \u00a0phosphatase,\t\r \u00a0SopB,\t\r \u00a0localizes\t\r \u00a0to\t\r \u00a0PtdIns(3)P-\u00ad\u2010containing\t\r \u00a0endosomes\t\r \u00a0and\t\r \u00a0perturbs\t\r \u00a0normal\t\r \u00a0endosome\t\r \u00a0to\t\r \u00a0lysosome\t\r \u00a0trafficking.\t\r \u00a0 Biochemical\t\r \u00a0Journal\t\r \u00a0395,\t\r \u00a0239-\u00ad\u2010247\t\r \u00a0(2006).\t\r \u00a0472.\t\r \u00a0 R.\t\r \u00a0M.\t\r \u00a0Gibson,\t\r \u00a0A.\t\r \u00a0L.\t\r \u00a0Wilson-\u00ad\u2010Delfosse,\t\r \u00a0RhoGDI-\u00ad\u2010binding-\u00ad\u2010defective\t\r \u00a0mutant\t\r \u00a0of\t\r \u00a0Cdc42Hs\t\r \u00a0targets\t\r \u00a0to\t\r \u00a0membranes\t\r \u00a0and\t\r \u00a0activates\t\r \u00a0filopodia\t\r \u00a0formation\t\r \u00a0but\t\r \u00a0does\t\r \u00a0not\t\r \u00a0cycle\t\r \u00a0with\t\r \u00a0the\t\r \u00a0cytosol\t\r \u00a0of\t\r \u00a0mammalian\t\r \u00a0cells.\t\r \u00a0Biochem\t\r \u00a0J\t\r \u00a0359,\t\r \u00a0285-\u00ad\u2010294\t\r \u00a0(2001).\t\r \u00a0473.\t\r \u00a0 L.\t\r \u00a0A.\t\r \u00a0Marotti,\t\r \u00a0Jr.,\t\r \u00a0R.\t\r \u00a0Newitt,\t\r \u00a0Y.\t\r \u00a0Wang,\t\r \u00a0R.\t\r \u00a0Aebersold,\t\r \u00a0H.\t\r \u00a0G.\t\r \u00a0Dohlman,\t\r \u00a0Direct\t\r \u00a0identification\t\r \u00a0of\t\r \u00a0a\t\r \u00a0G\t\r \u00a0protein\t\r \u00a0ubiquitination\t\r \u00a0site\t\r \u00a0by\t\r \u00a0mass\t\r \u00a0spectrometry.\t\r \u00a0 Biochemistry\t\r \u00a041,\t\r \u00a05067-\u00ad\u20105074\t\r \u00a0(2002).\t\r \u00a0474.\t\r \u00a0 A.\t\r \u00a0Angot,\t\r \u00a0A.\t\r \u00a0Vergunst,\t\r \u00a0S.\t\r \u00a0Genin,\t\r \u00a0N.\t\r \u00a0Peeters,\t\r \u00a0Exploitation\t\r \u00a0of\t\r \u00a0eukaryotic\t\r \u00a0ubiquitin\t\r \u00a0signaling\t\r \u00a0pathways\t\r \u00a0by\t\r \u00a0effectors\t\r \u00a0translocated\t\r \u00a0by\t\r \u00a0bacterial\t\r \u00a0type\t\r \u00a0III\t\r \u00a0and\t\r \u00a0type\t\r \u00a0IV\t\r \u00a0secretion\t\r \u00a0systems.\t\r \u00a0PLoS\t\r \u00a0Pathog\t\r \u00a03,\t\r \u00a0e3\t\r \u00a0(2007).\t\r \u00a0475.\t\r \u00a0 Y.\t\r \u00a0Mosesson,\t\r \u00a0Y.\t\r \u00a0Yarden,\t\r \u00a0Monoubiquitylation:\t\r \u00a0a\t\r \u00a0recurrent\t\r \u00a0theme\t\r \u00a0in\t\r \u00a0membrane\t\r \u00a0protein\t\r \u00a0transport.\t\r \u00a0Isr\t\r \u00a0Med\t\r \u00a0Assoc\t\r \u00a0J\t\r \u00a08,\t\r \u00a0233-\u00ad\u2010237\t\r \u00a0(2006).\t\r \u00a0476.\t\r \u00a0 L.\t\r \u00a0Hicke,\t\r \u00a0R.\t\r \u00a0Dunn,\t\r \u00a0Regulation\t\r \u00a0of\t\r \u00a0membrane\t\r \u00a0protein\t\r \u00a0transport\t\r \u00a0by\t\r \u00a0ubiquitin\t\r \u00a0and\t\r \u00a0ubiquitin-\u00ad\u2010binding\t\r \u00a0proteins.\t\r \u00a0Annu\t\r \u00a0Rev\t\r \u00a0Cell\t\r \u00a0Dev\t\r \u00a0Biol\t\r \u00a019,\t\r \u00a0141-\u00ad\u2010172\t\r \u00a0(2003).\t\r \u00a0477.\t\r \u00a0 D.\t\r \u00a0Mukhopadhyay,\t\r \u00a0H.\t\r \u00a0Riezman,\t\r \u00a0Proteasome-\u00ad\u2010independent\t\r \u00a0functions\t\r \u00a0of\t\r \u00a0ubiquitin\t\r \u00a0in\t\r \u00a0endocytosis\t\r \u00a0and\t\r \u00a0signaling.\t\r \u00a0Science\t\r \u00a0315,\t\r \u00a0201-\u00ad\u2010205\t\r \u00a0(2007).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 179\t\r \u00a0 478.\t\r \u00a0 M.\t\r \u00a0D.\t\r \u00a0Petroski,\t\r \u00a0R.\t\r \u00a0J.\t\r \u00a0Deshaies,\t\r \u00a0Context\t\r \u00a0of\t\r \u00a0multiubiquitin\t\r \u00a0chain\t\r \u00a0attachment\t\r \u00a0influences\t\r \u00a0the\t\r \u00a0rate\t\r \u00a0of\t\r \u00a0Sic1\t\r \u00a0degradation.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a011,\t\r \u00a01435-\u00ad\u20101444\t\r \u00a0(2003).\t\r \u00a0479.\t\r \u00a0 L.\t\r \u00a0D.\t\r \u00a0Rogers,\t\r \u00a0L.\t\r \u00a0J.\t\r \u00a0Foster,\t\r \u00a0The\t\r \u00a0dynamic\t\r \u00a0phagosome\t\r \u00a0proteome\t\r \u00a0and\t\r \u00a0the\t\r \u00a0contribution\t\r \u00a0of\t\r \u00a0the\t\r \u00a0ER.\t\r \u00a0Proceedings\t\r \u00a0of\t\r \u00a0the\t\r \u00a0National\t\r \u00a0Academy\t\r \u00a0of\t\r \u00a0Sciences\t\r \u00a0104,\t\r \u00a018520-\u00ad\u201018525\t\r \u00a0(2007).\t\r \u00a0480.\t\r \u00a0 J.\t\r \u00a0D.\t\r \u00a0Graves,\t\r \u00a0E.\t\r \u00a0G.\t\r \u00a0Krebs,\t\r \u00a0Protein\t\r \u00a0phosphorylation\t\r \u00a0and\t\r \u00a0signal\t\r \u00a0transduction.\t\r \u00a0 Pharmacol\t\r \u00a0Ther\t\r \u00a082,\t\r \u00a0111-\u00ad\u2010121\t\r \u00a0(1999).\t\r \u00a0481.\t\r \u00a0 H.\t\r \u00a0Issaq,\t\r \u00a0T.\t\r \u00a0Veenstra,\t\r \u00a0Two-\u00ad\u2010dimensional\t\r \u00a0polyacrylamide\t\r \u00a0gel\t\r \u00a0electrophoresis\t\r \u00a0(2D-\u00ad\u2010PAGE):\t\r \u00a0advances\t\r \u00a0and\t\r \u00a0perspectives.\t\r \u00a0Biotechniques\t\r \u00a044,\t\r \u00a0697-\u00ad\u2010698,\t\r \u00a0700\t\r \u00a0(2008).\t\r \u00a0482.\t\r \u00a0 T.\t\r \u00a0E.\t\r \u00a0Thingholm,\t\r \u00a0O.\t\r \u00a0N.\t\r \u00a0Jensen,\t\r \u00a0M.\t\r \u00a0R.\t\r \u00a0Larsen,\t\r \u00a0Analytical\t\r \u00a0strategies\t\r \u00a0for\t\r \u00a0phosphoproteomics.\t\r \u00a0Proteomics\t\r \u00a09,\t\r \u00a01451-\u00ad\u20101468\t\r \u00a0(2009).\t\r \u00a0483.\t\r \u00a0 S.\t\r \u00a0Barik,\t\r \u00a0Protein\t\r \u00a0phosphorylation\t\r \u00a0and\t\r \u00a0signal\t\r \u00a0transduction.\t\r \u00a0Subcell\t\r \u00a0Biochem\t\r \u00a0 26,\t\r \u00a0115-\u00ad\u2010164\t\r \u00a0(1996).\t\r \u00a0484.\t\r \u00a0 G.\t\r \u00a0T.\t\r \u00a0Cantin,\t\r \u00a0J.\t\r \u00a0D.\t\r \u00a0Venable,\t\r \u00a0D.\t\r \u00a0Cociorva,\t\r \u00a0J.\t\r \u00a0R.\t\r \u00a0Yates,\t\r \u00a03rd,\t\r \u00a0Quantitative\t\r \u00a0phosphoproteomic\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0the\t\r \u00a0tumor\t\r \u00a0necrosis\t\r \u00a0factor\t\r \u00a0pathway.\t\r \u00a0J\t\r \u00a0Proteome\t\r \u00a0 Res\t\r \u00a05,\t\r \u00a0127-\u00ad\u2010134\t\r \u00a0(2006).\t\r \u00a0485.\t\r \u00a0 M.\t\r \u00a0Kruger,\t\r \u00a0I.\t\r \u00a0Kratchmarova,\t\r \u00a0B.\t\r \u00a0Blagoev,\t\r \u00a0Y.\t\r \u00a0H.\t\r \u00a0Tseng,\t\r \u00a0C.\t\r \u00a0R.\t\r \u00a0Kahn,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Dissection\t\r \u00a0of\t\r \u00a0the\t\r \u00a0insulin\t\r \u00a0signaling\t\r \u00a0pathway\t\r \u00a0via\t\r \u00a0quantitative\t\r \u00a0phosphoproteomics.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a0105,\t\r \u00a02451-\u00ad\u20102456\t\r \u00a0(2008).\t\r \u00a0486.\t\r \u00a0 Y.\t\r \u00a0Zhang,\t\r \u00a0A.\t\r \u00a0Wolf-\u00ad\u2010Yadlin,\t\r \u00a0P.\t\r \u00a0L.\t\r \u00a0Ross,\t\r \u00a0D.\t\r \u00a0J.\t\r \u00a0Pappin,\t\r \u00a0J.\t\r \u00a0Rush,\t\r \u00a0D.\t\r \u00a0A.\t\r \u00a0Lauffenburger,\t\r \u00a0F.\t\r \u00a0M.\t\r \u00a0White,\t\r \u00a0Time-\u00ad\u2010resolved\t\r \u00a0mass\t\r \u00a0spectrometry\t\r \u00a0of\t\r \u00a0tyrosine\t\r \u00a0phosphorylation\t\r \u00a0sites\t\r \u00a0in\t\r \u00a0the\t\r \u00a0epidermal\t\r \u00a0growth\t\r \u00a0factor\t\r \u00a0receptor\t\r \u00a0signaling\t\r \u00a0network\t\r \u00a0reveals\t\r \u00a0dynamic\t\r \u00a0modules.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Proteomics\t\r \u00a04,\t\r \u00a01240-\u00ad\u20101250\t\r \u00a0(2005).\t\r \u00a0487.\t\r \u00a0 M.\t\r \u00a0Oyama,\t\r \u00a0H.\t\r \u00a0Kozuka-\u00ad\u2010Hata,\t\r \u00a0S.\t\r \u00a0Tasaki,\t\r \u00a0K.\t\r \u00a0Semba,\t\r \u00a0S.\t\r \u00a0Hattori,\t\r \u00a0S.\t\r \u00a0Sugano,\t\r \u00a0J.\t\r \u00a0Inoue,\t\r \u00a0T.\t\r \u00a0Yamamoto,\t\r \u00a0Temporal\t\r \u00a0perturbation\t\r \u00a0of\t\r \u00a0tyrosine\t\r \u00a0phosphoproteome\t\r \u00a0dynamics\t\r \u00a0reveals\t\r \u00a0the\t\r \u00a0system-\u00ad\u2010wide\t\r \u00a0regulatory\t\r \u00a0networks.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Proteomics\t\r \u00a0 8,\t\r \u00a0226-\u00ad\u2010231\t\r \u00a0(2009).\t\r \u00a0488.\t\r \u00a0 P.\t\r \u00a0J.\t\r \u00a0Boersema,\t\r \u00a0S.\t\r \u00a0Mohammed,\t\r \u00a0A.\t\r \u00a0J.\t\r \u00a0Heck,\t\r \u00a0Phosphopeptide\t\r \u00a0fragmentation\t\r \u00a0and\t\r \u00a0analysis\t\r \u00a0by\t\r \u00a0mass\t\r \u00a0spectrometry.\t\r \u00a0J\t\r \u00a0Mass\t\r \u00a0Spectrom\t\r \u00a044,\t\r \u00a0861-\u00ad\u2010878\t\r \u00a0(2009).\t\r \u00a0489.\t\r \u00a0 L.\t\r \u00a0D.\t\r \u00a0Rogers,\t\r \u00a0L.\t\r \u00a0J.\t\r \u00a0Foster,\t\r \u00a0Phosphoproteomics-\u00ad\u2010-\u00ad\u2010finally\t\r \u00a0fulfilling\t\r \u00a0the\t\r \u00a0promise?\t\r \u00a0 Mol\t\r \u00a0Biosyst\t\r \u00a05,\t\r \u00a01122-\u00ad\u20101129\t\r \u00a0(2009).\t\r \u00a0490.\t\r \u00a0 J.\t\r \u00a0Rappsilber,\t\r \u00a0Y.\t\r \u00a0Ishihama,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Stop\t\r \u00a0and\t\r \u00a0go\t\r \u00a0extraction\t\r \u00a0tips\t\r \u00a0for\t\r \u00a0matrix-\u00ad\u2010assisted\t\r \u00a0laser\t\r \u00a0desorption\/ionization,\t\r \u00a0nanoelectrospray,\t\r \u00a0and\t\r \u00a0LC\/MS\t\r \u00a0sample\t\r \u00a0pretreatment\t\r \u00a0in\t\r \u00a0proteomics.\t\r \u00a0Anal\t\r \u00a0Chem\t\r \u00a075,\t\r \u00a0663-\u00ad\u2010670\t\r \u00a0(2003).\t\r \u00a0491.\t\r \u00a0 L.\t\r \u00a0L.\t\r \u00a0Manza,\t\r \u00a0S.\t\r \u00a0L.\t\r \u00a0Stamer,\t\r \u00a0A.\t\r \u00a0J.\t\r \u00a0Ham,\t\r \u00a0S.\t\r \u00a0G.\t\r \u00a0Codreanu,\t\r \u00a0D.\t\r \u00a0C.\t\r \u00a0Liebler,\t\r \u00a0Sample\t\r \u00a0preparation\t\r \u00a0and\t\r \u00a0digestion\t\r \u00a0for\t\r \u00a0proteomic\t\r \u00a0analyses\t\r \u00a0using\t\r \u00a0spin\t\r \u00a0filters.\t\r \u00a0 Proteomics\t\r \u00a05,\t\r \u00a01742-\u00ad\u20101745\t\r \u00a0(2005).\t\r \u00a0492.\t\r \u00a0 J.\t\r \u00a0R.\t\r \u00a0Wisniewski,\t\r \u00a0A.\t\r \u00a0Zougman,\t\r \u00a0N.\t\r \u00a0Nagaraj,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Universal\t\r \u00a0sample\t\r \u00a0preparation\t\r \u00a0method\t\r \u00a0for\t\r \u00a0proteome\t\r \u00a0analysis.\t\r \u00a0Nat\t\r \u00a0Methods\t\r \u00a06,\t\r \u00a0359-\u00ad\u2010362\t\r \u00a0(2009).\t\r \u00a0493.\t\r \u00a0 J.\t\r \u00a0Zhou,\t\r \u00a0T.\t\r \u00a0Zhou,\t\r \u00a0R.\t\r \u00a0Cao,\t\r \u00a0Z.\t\r \u00a0Liu,\t\r \u00a0J.\t\r \u00a0Shen,\t\r \u00a0P.\t\r \u00a0Chen,\t\r \u00a0X.\t\r \u00a0Wang,\t\r \u00a0S.\t\r \u00a0Liang,\t\r \u00a0Evaluation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0application\t\r \u00a0of\t\r \u00a0sodium\t\r \u00a0deoxycholate\t\r \u00a0to\t\r \u00a0proteomic\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0rat\t\r \u00a0hippocampal\t\r \u00a0plasma\t\r \u00a0membrane.\t\r \u00a0J\t\r \u00a0Proteome\t\r \u00a0Res\t\r \u00a05,\t\r \u00a02547-\u00ad\u20102553\t\r \u00a0(2006).\t\r \u00a0494.\t\r \u00a0 T.\t\r \u00a0Masuda,\t\r \u00a0M.\t\r \u00a0Tomita,\t\r \u00a0Y.\t\r \u00a0Ishihama,\t\r \u00a0Phase\t\r \u00a0transfer\t\r \u00a0surfactant-\u00ad\u2010aided\t\r \u00a0trypsin\t\r \u00a0digestion\t\r \u00a0for\t\r \u00a0membrane\t\r \u00a0proteome\t\r \u00a0analysis.\t\r \u00a0J\t\r \u00a0Proteome\t\r \u00a0Res\t\r \u00a07,\t\r \u00a0731-\u00ad\u2010740\t\r \u00a0(2008).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 180\t\r \u00a0 495.\t\r \u00a0 J.\t\r \u00a0Villen,\t\r \u00a0S.\t\r \u00a0A.\t\r \u00a0Beausoleil,\t\r \u00a0S.\t\r \u00a0A.\t\r \u00a0Gerber,\t\r \u00a0S.\t\r \u00a0P.\t\r \u00a0Gygi,\t\r \u00a0Large-\u00ad\u2010scale\t\r \u00a0phosphorylation\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0mouse\t\r \u00a0liver.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a0104,\t\r \u00a01488-\u00ad\u20101493\t\r \u00a0(2007).\t\r \u00a0496.\t\r \u00a0 J.\t\r \u00a0V.\t\r \u00a0Olsen,\t\r \u00a0M.\t\r \u00a0Mann,\t\r \u00a0Improved\t\r \u00a0peptide\t\r \u00a0identification\t\r \u00a0in\t\r \u00a0proteomics\t\r \u00a0by\t\r \u00a0two\t\r \u00a0consecutive\t\r \u00a0stages\t\r \u00a0of\t\r \u00a0mass\t\r \u00a0spectrometric\t\r \u00a0fragmentation.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0 S\t\r \u00a0A\t\r \u00a0101,\t\r \u00a013417-\u00ad\u201013422\t\r \u00a0(2004).\t\r \u00a0497.\t\r \u00a0 G.\t\r \u00a0R.\t\r \u00a0Stark,\t\r \u00a0Reactions\t\r \u00a0of\t\r \u00a0cyanate\t\r \u00a0with\t\r \u00a0functional\t\r \u00a0groups\t\r \u00a0of\t\r \u00a0proteins.\t\r \u00a0IV.\t\r \u00a0Inertness\t\r \u00a0of\t\r \u00a0aliphatic\t\r \u00a0hydroxyl\t\r \u00a0groups.\t\r \u00a0Formation\t\r \u00a0of\t\r \u00a0carbamyl-\u00ad\u2010\t\r \u00a0and\t\r \u00a0acylhydantoins.\t\r \u00a0Biochemistry\t\r \u00a04,\t\r \u00a02363-\u00ad\u20102367\t\r \u00a0(1965).\t\r \u00a0498.\t\r \u00a0 G.\t\r \u00a0R.\t\r \u00a0Stark,\t\r \u00a0Reactions\t\r \u00a0of\t\r \u00a0cyanate\t\r \u00a0with\t\r \u00a0functional\t\r \u00a0groups\t\r \u00a0of\t\r \u00a0proteins.\t\r \u00a03.\t\r \u00a0Reactions\t\r \u00a0with\t\r \u00a0amino\t\r \u00a0and\t\r \u00a0carboxyl\t\r \u00a0groups.\t\r \u00a0Biochemistry\t\r \u00a04,\t\r \u00a01030-\u00ad\u20101036\t\r \u00a0(1965).\t\r \u00a0499.\t\r \u00a0 N.\t\r \u00a0L.\t\r \u00a0Anderson,\t\r \u00a0N.\t\r \u00a0G.\t\r \u00a0Anderson,\t\r \u00a0The\t\r \u00a0human\t\r \u00a0plasma\t\r \u00a0proteome:\t\r \u00a0history,\t\r \u00a0character,\t\r \u00a0and\t\r \u00a0diagnostic\t\r \u00a0prospects.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Proteomics\t\r \u00a01,\t\r \u00a0845-\u00ad\u2010867\t\r \u00a0(2002).\t\r \u00a0500.\t\r \u00a0 J.\t\r \u00a0R.\t\r \u00a0Yates,\t\r \u00a0C.\t\r \u00a0I.\t\r \u00a0Ruse,\t\r \u00a0A.\t\r \u00a0Nakorchevsky,\t\r \u00a0Proteomics\t\r \u00a0by\t\r \u00a0mass\t\r \u00a0spectrometry:\t\r \u00a0approaches,\t\r \u00a0advances,\t\r \u00a0and\t\r \u00a0applications.\t\r \u00a0Annu\t\r \u00a0Rev\t\r \u00a0Biomed\t\r \u00a0Eng\t\r \u00a011,\t\r \u00a049-\u00ad\u201079\t\r \u00a0(2009).\t\r \u00a0501.\t\r \u00a0 J.\t\r \u00a0J.\t\r \u00a0Coon,\t\r \u00a0J.\t\r \u00a0Shabanowitz,\t\r \u00a0D.\t\r \u00a0F.\t\r \u00a0Hunt,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Syka,\t\r \u00a0Electron\t\r \u00a0transfer\t\r \u00a0dissociation\t\r \u00a0of\t\r \u00a0peptide\t\r \u00a0anions.\t\r \u00a0J\t\r \u00a0Am\t\r \u00a0Soc\t\r \u00a0Mass\t\r \u00a0Spectrom\t\r \u00a016,\t\r \u00a0880-\u00ad\u2010882\t\r \u00a0(2005).\t\r \u00a0502.\t\r \u00a0 J.\t\r \u00a0E.\t\r \u00a0Syka,\t\r \u00a0J.\t\r \u00a0J.\t\r \u00a0Coon,\t\r \u00a0M.\t\r \u00a0J.\t\r \u00a0Schroeder,\t\r \u00a0J.\t\r \u00a0Shabanowitz,\t\r \u00a0D.\t\r \u00a0F.\t\r \u00a0Hunt,\t\r \u00a0Peptide\t\r \u00a0and\t\r \u00a0protein\t\r \u00a0sequence\t\r \u00a0analysis\t\r \u00a0by\t\r \u00a0electron\t\r \u00a0transfer\t\r \u00a0dissociation\t\r \u00a0mass\t\r \u00a0spectrometry.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a0101,\t\r \u00a09528-\u00ad\u20109533\t\r \u00a0(2004).\t\r \u00a0503.\t\r \u00a0 M.\t\r \u00a0A.\t\r \u00a0Bakowski,\t\r \u00a0V.\t\r \u00a0Braun,\t\r \u00a0J.\t\r \u00a0H.\t\r \u00a0Brumell,\t\r \u00a0Salmonella-\u00ad\u2010containing\t\r \u00a0vacuoles:\t\r \u00a0directing\t\r \u00a0traffic\t\r \u00a0and\t\r \u00a0nesting\t\r \u00a0to\t\r \u00a0grow.\t\r \u00a0Traffic\t\r \u00a09,\t\r \u00a02022-\u00ad\u20102031\t\r \u00a0(2008).\t\r \u00a0504.\t\r \u00a0 L.\t\r \u00a0D.\t\r \u00a0Rogers,\t\r \u00a0Y.\t\r \u00a0Fang,\t\r \u00a0L.\t\r \u00a0J.\t\r \u00a0Foster,\t\r \u00a0An\t\r \u00a0integrated\t\r \u00a0global\t\r \u00a0strategy\t\r \u00a0for\t\r \u00a0cell\t\r \u00a0lysis,\t\r \u00a0fractionation,\t\r \u00a0enrichment\t\r \u00a0and\t\r \u00a0mass\t\r \u00a0spectrometric\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0phosphorylated\t\r \u00a0peptides.\t\r \u00a0Mol\t\r \u00a0Biosyst\t\r \u00a06,\t\r \u00a0822-\u00ad\u2010829\t\r \u00a0(2010).\t\r \u00a0505.\t\r \u00a0 M.\t\r \u00a0E.\t\r \u00a0Futschik,\t\r \u00a0B.\t\r \u00a0Carlisle,\t\r \u00a0Noise-\u00ad\u2010robust\t\r \u00a0soft\t\r \u00a0clustering\t\r \u00a0of\t\r \u00a0gene\t\r \u00a0expression\t\r \u00a0time-\u00ad\u2010course\t\r \u00a0data.\t\r \u00a0J\t\r \u00a0Bioinform\t\r \u00a0Comput\t\r \u00a0Biol\t\r \u00a03,\t\r \u00a0965-\u00ad\u2010988\t\r \u00a0(2005).\t\r \u00a0506.\t\r \u00a0 G.\t\r \u00a0Dennis,\t\r \u00a0Jr.,\t\r \u00a0B.\t\r \u00a0T.\t\r \u00a0Sherman,\t\r \u00a0D.\t\r \u00a0A.\t\r \u00a0Hosack,\t\r \u00a0J.\t\r \u00a0Yang,\t\r \u00a0W.\t\r \u00a0Gao,\t\r \u00a0H.\t\r \u00a0C.\t\r \u00a0Lane,\t\r \u00a0R.\t\r \u00a0A.\t\r \u00a0Lempicki,\t\r \u00a0DAVID:\t\r \u00a0Database\t\r \u00a0for\t\r \u00a0Annotation,\t\r \u00a0Visualization,\t\r \u00a0and\t\r \u00a0Integrated\t\r \u00a0Discovery.\t\r \u00a0Genome\t\r \u00a0Biol\t\r \u00a04,\t\r \u00a0P3\t\r \u00a0(2003).\t\r \u00a0507.\t\r \u00a0 W.\t\r \u00a0Huang\t\r \u00a0da,\t\r \u00a0B.\t\r \u00a0T.\t\r \u00a0Sherman,\t\r \u00a0R.\t\r \u00a0A.\t\r \u00a0Lempicki,\t\r \u00a0Systematic\t\r \u00a0and\t\r \u00a0integrative\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0large\t\r \u00a0gene\t\r \u00a0lists\t\r \u00a0using\t\r \u00a0DAVID\t\r \u00a0bioinformatics\t\r \u00a0resources.\t\r \u00a0Nat\t\r \u00a0Protoc\t\r \u00a0 4,\t\r \u00a044-\u00ad\u201057\t\r \u00a0(2009).\t\r \u00a0508.\t\r \u00a0 D.\t\r \u00a0Schwartz,\t\r \u00a0S.\t\r \u00a0P.\t\r \u00a0Gygi,\t\r \u00a0An\t\r \u00a0iterative\t\r \u00a0statistical\t\r \u00a0approach\t\r \u00a0to\t\r \u00a0the\t\r \u00a0identification\t\r \u00a0of\t\r \u00a0protein\t\r \u00a0phosphorylation\t\r \u00a0motifs\t\r \u00a0from\t\r \u00a0large-\u00ad\u2010scale\t\r \u00a0data\t\r \u00a0sets.\t\r \u00a0Nat\t\r \u00a0Biotechnol\t\r \u00a023,\t\r \u00a01391-\u00ad\u20101398\t\r \u00a0(2005).\t\r \u00a0509.\t\r \u00a0 S.\t\r \u00a0Pelech,\t\r \u00a0C.\t\r \u00a0Sutter,\t\r \u00a0H.\t\r \u00a0Zhang,\t\r \u00a0Kinetworks\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a0multiblot\t\r \u00a0analysis.\t\r \u00a0 Methods\t\r \u00a0Mol\t\r \u00a0Biol\t\r \u00a0218,\t\r \u00a099-\u00ad\u2010111\t\r \u00a0(2003).\t\r \u00a0510.\t\r \u00a0 M.\t\r \u00a0C.\t\r \u00a0Schlumberger,\t\r \u00a0A.\t\r \u00a0J.\t\r \u00a0Muller,\t\r \u00a0K.\t\r \u00a0Ehrbar,\t\r \u00a0B.\t\r \u00a0Winnen,\t\r \u00a0I.\t\r \u00a0Duss,\t\r \u00a0B.\t\r \u00a0Stecher,\t\r \u00a0W.\t\r \u00a0D.\t\r \u00a0Hardt,\t\r \u00a0Real-\u00ad\u2010time\t\r \u00a0imaging\t\r \u00a0of\t\r \u00a0type\t\r \u00a0III\t\r \u00a0secretion:\t\r \u00a0Salmonella\t\r \u00a0SipA\t\r \u00a0injection\t\r \u00a0into\t\r \u00a0host\t\r \u00a0cells.\t\r \u00a0Proc\t\r \u00a0Natl\t\r \u00a0Acad\t\r \u00a0Sci\t\r \u00a0U\t\r \u00a0S\t\r \u00a0A\t\r \u00a0102,\t\r \u00a012548-\u00ad\u201012553\t\r \u00a0(2005).\t\r \u00a0511.\t\r \u00a0 W.\t\r \u00a0F.\t\r \u00a0Waas,\t\r \u00a0H.\t\r \u00a0H.\t\r \u00a0Lo,\t\r \u00a0K.\t\r \u00a0N.\t\r \u00a0Dalby,\t\r \u00a0The\t\r \u00a0kinetic\t\r \u00a0mechanism\t\r \u00a0of\t\r \u00a0the\t\r \u00a0dual\t\r \u00a0phosphorylation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0ATF2\t\r \u00a0transcription\t\r \u00a0factor\t\r \u00a0by\t\r \u00a0p38\t\r \u00a0mitogen-\u00ad\u2010activated\t\r \u00a0protein\t\r \u00a0(MAP)\t\r \u00a0kinase\t\r \u00a0alpha.\t\r \u00a0Implications\t\r \u00a0for\t\r \u00a0signal\/response\t\r \u00a0profiles\t\r \u00a0of\t\r \u00a0MAP\t\r \u00a0kinase\t\r \u00a0pathways.\t\r \u00a0J\t\r \u00a0Biol\t\r \u00a0Chem\t\r \u00a0276,\t\r \u00a05676-\u00ad\u20105684\t\r \u00a0(2001).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 181\t\r \u00a0 512.\t\r \u00a0 B.\t\r \u00a0Camuzeaux,\t\r \u00a0J.\t\r \u00a0Diring,\t\r \u00a0P.\t\r \u00a0J.\t\r \u00a0Hamard,\t\r \u00a0M.\t\r \u00a0Oulad-\u00ad\u2010Abdelghani,\t\r \u00a0M.\t\r \u00a0Donzeau,\t\r \u00a0M.\t\r \u00a0Vigneron,\t\r \u00a0C.\t\r \u00a0Kedinger,\t\r \u00a0B.\t\r \u00a0Chatton,\t\r \u00a0p38beta2-\u00ad\u2010mediated\t\r \u00a0phosphorylation\t\r \u00a0and\t\r \u00a0sumoylation\t\r \u00a0of\t\r \u00a0ATF7\t\r \u00a0are\t\r \u00a0mutually\t\r \u00a0exclusive.\t\r \u00a0J\t\r \u00a0Mol\t\r \u00a0Biol\t\r \u00a0384,\t\r \u00a0980-\u00ad\u2010991\t\r \u00a0(2008).\t\r \u00a0513.\t\r \u00a0 L.\t\r \u00a0M.\t\r \u00a0Chen,\t\r \u00a0S.\t\r \u00a0Bagrodia,\t\r \u00a0R.\t\r \u00a0A.\t\r \u00a0Cerione,\t\r \u00a0J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0Requirement\t\r \u00a0of\t\r \u00a0p21-\u00ad\u2010activated\t\r \u00a0kinase\t\r \u00a0(PAK)\t\r \u00a0for\t\r \u00a0Salmonella\t\r \u00a0typhimurium-\u00ad\u2010induced\t\r \u00a0nuclear\t\r \u00a0responses.\t\r \u00a0J\t\r \u00a0Exp\t\r \u00a0Med\t\r \u00a0189,\t\r \u00a01479-\u00ad\u20101488\t\r \u00a0(1999).\t\r \u00a0514.\t\r \u00a0 M.\t\r \u00a0J.\t\r \u00a0Pallen,\t\r \u00a0S.\t\r \u00a0A.\t\r \u00a0Beatson,\t\r \u00a0C.\t\r \u00a0M.\t\r \u00a0Bailey,\t\r \u00a0Bioinformatics,\t\r \u00a0genomics\t\r \u00a0and\t\r \u00a0evolution\t\r \u00a0of\t\r \u00a0non-\u00ad\u2010flagellar\t\r \u00a0type-\u00ad\u2010III\t\r \u00a0secretion\t\r \u00a0systems:\t\r \u00a0a\t\r \u00a0Darwinian\t\r \u00a0perspective.\t\r \u00a0FEMS\t\r \u00a0Microbiol\t\r \u00a0Rev\t\r \u00a029,\t\r \u00a0201-\u00ad\u2010229\t\r \u00a0(2005).\t\r \u00a0515.\t\r \u00a0 O.\t\r \u00a0Shibolet,\t\r \u00a0D.\t\r \u00a0K.\t\r \u00a0Podolsky,\t\r \u00a0TLRs\t\r \u00a0in\t\r \u00a0the\t\r \u00a0Gut.\t\r \u00a0IV.\t\r \u00a0Negative\t\r \u00a0regulation\t\r \u00a0of\t\r \u00a0Toll-\u00ad\u2010like\t\r \u00a0receptors\t\r \u00a0and\t\r \u00a0intestinal\t\r \u00a0homeostasis:\t\r \u00a0addition\t\r \u00a0by\t\r \u00a0subtraction.\t\r \u00a0Am\t\r \u00a0J\t\r \u00a0 Physiol\t\r \u00a0Gastrointest\t\r \u00a0Liver\t\r \u00a0Physiol\t\r \u00a0292,\t\r \u00a0G1469-\u00ad\u20101473\t\r \u00a0(2007).\t\r \u00a0516.\t\r \u00a0 K.\t\r \u00a0Grohmanova,\t\r \u00a0D.\t\r \u00a0Schlaepfer,\t\r \u00a0D.\t\r \u00a0Hess,\t\r \u00a0P.\t\r \u00a0Gutierrez,\t\r \u00a0M.\t\r \u00a0Beck,\t\r \u00a0R.\t\r \u00a0Kroschewski,\t\r \u00a0Phosphorylation\t\r \u00a0of\t\r \u00a0IQGAP1\t\r \u00a0modulates\t\r \u00a0its\t\r \u00a0binding\t\r \u00a0to\t\r \u00a0Cdc42,\t\r \u00a0revealing\t\r \u00a0a\t\r \u00a0new\t\r \u00a0type\t\r \u00a0of\t\r \u00a0rho-\u00ad\u2010GTPase\t\r \u00a0regulator.\t\r \u00a0J\t\r \u00a0Biol\t\r \u00a0Chem\t\r \u00a0279,\t\r \u00a048495-\u00ad\u201048504\t\r \u00a0(2004).\t\r \u00a0517.\t\r \u00a0 J.\t\r \u00a0Yuan,\t\r \u00a0O.\t\r \u00a0Rey,\t\r \u00a0E.\t\r \u00a0Rozengurt,\t\r \u00a0Activation\t\r \u00a0of\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a0D3\t\r \u00a0by\t\r \u00a0signaling\t\r \u00a0through\t\r \u00a0Rac\t\r \u00a0and\t\r \u00a0the\t\r \u00a0alpha\t\r \u00a0subunits\t\r \u00a0of\t\r \u00a0the\t\r \u00a0heterotrimeric\t\r \u00a0G\t\r \u00a0proteins\t\r \u00a0G12\t\r \u00a0and\t\r \u00a0G13.\t\r \u00a0Cell\t\r \u00a0Signal\t\r \u00a018,\t\r \u00a01051-\u00ad\u20101062\t\r \u00a0(2006).\t\r \u00a0518.\t\r \u00a0 M.\t\r \u00a0K.\t\r \u00a0Pflum,\t\r \u00a0J.\t\r \u00a0K.\t\r \u00a0Tong,\t\r \u00a0W.\t\r \u00a0S.\t\r \u00a0Lane,\t\r \u00a0S.\t\r \u00a0L.\t\r \u00a0Schreiber,\t\r \u00a0Histone\t\r \u00a0deacetylase\t\r \u00a01\t\r \u00a0phosphorylation\t\r \u00a0promotes\t\r \u00a0enzymatic\t\r \u00a0activity\t\r \u00a0and\t\r \u00a0complex\t\r \u00a0formation.\t\r \u00a0J\t\r \u00a0Biol\t\r \u00a0 Chem\t\r \u00a0276,\t\r \u00a047733-\u00ad\u201047741\t\r \u00a0(2001).\t\r \u00a0519.\t\r \u00a0 X.\t\r \u00a0G.\t\r \u00a0Jiang,\t\r \u00a0Y.\t\r \u00a0Wang,\t\r \u00a0Phosphorylation\t\r \u00a0of\t\r \u00a0human\t\r \u00a0high\t\r \u00a0mobility\t\r \u00a0group\t\r \u00a0N1\t\r \u00a0protein\t\r \u00a0by\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a0CK2.\t\r \u00a0Biochem\t\r \u00a0Biophys\t\r \u00a0Res\t\r \u00a0Commun\t\r \u00a0345,\t\r \u00a01497-\u00ad\u20101503\t\r \u00a0(2006).\t\r \u00a0520.\t\r \u00a0 L.\t\r \u00a0del\t\r \u00a0Peso,\t\r \u00a0M.\t\r \u00a0Gonzalez-\u00ad\u2010Garcia,\t\r \u00a0C.\t\r \u00a0Page,\t\r \u00a0R.\t\r \u00a0Herrera,\t\r \u00a0G.\t\r \u00a0Nunez,\t\r \u00a0Interleukin-\u00ad\u20103-\u00ad\u2010induced\t\r \u00a0phosphorylation\t\r \u00a0of\t\r \u00a0BAD\t\r \u00a0through\t\r \u00a0the\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a0Akt.\t\r \u00a0Science\t\r \u00a0278,\t\r \u00a0687-\u00ad\u2010689\t\r \u00a0(1997).\t\r \u00a0521.\t\r \u00a0 S.\t\r \u00a0R.\t\r \u00a0Datta,\t\r \u00a0H.\t\r \u00a0Dudek,\t\r \u00a0X.\t\r \u00a0Tao,\t\r \u00a0S.\t\r \u00a0Masters,\t\r \u00a0H.\t\r \u00a0Fu,\t\r \u00a0Y.\t\r \u00a0Gotoh,\t\r \u00a0M.\t\r \u00a0E.\t\r \u00a0Greenberg,\t\r \u00a0Akt\t\r \u00a0phosphorylation\t\r \u00a0of\t\r \u00a0BAD\t\r \u00a0couples\t\r \u00a0survival\t\r \u00a0signals\t\r \u00a0to\t\r \u00a0the\t\r \u00a0cell-\u00ad\u2010intrinsic\t\r \u00a0death\t\r \u00a0machinery.\t\r \u00a0Cell\t\r \u00a091,\t\r \u00a0231-\u00ad\u2010241\t\r \u00a0(1997).\t\r \u00a0522.\t\r \u00a0 A.\t\r \u00a0Gohla,\t\r \u00a0G.\t\r \u00a0M.\t\r \u00a0Bokoch,\t\r \u00a014-\u00ad\u20103-\u00ad\u20103\t\r \u00a0regulates\t\r \u00a0actin\t\r \u00a0dynamics\t\r \u00a0by\t\r \u00a0stabilizing\t\r \u00a0phosphorylated\t\r \u00a0cofilin.\t\r \u00a0Curr\t\r \u00a0Biol\t\r \u00a012,\t\r \u00a01704-\u00ad\u20101710\t\r \u00a0(2002).\t\r \u00a0523.\t\r \u00a0 D.\t\r \u00a0C.\t\r \u00a0Edwards,\t\r \u00a0L.\t\r \u00a0C.\t\r \u00a0Sanders,\t\r \u00a0G.\t\r \u00a0M.\t\r \u00a0Bokoch,\t\r \u00a0G.\t\r \u00a0N.\t\r \u00a0Gill,\t\r \u00a0Activation\t\r \u00a0of\t\r \u00a0LIM-\u00ad\u2010kinase\t\r \u00a0by\t\r \u00a0Pak1\t\r \u00a0couples\t\r \u00a0Rac\/Cdc42\t\r \u00a0GTPase\t\r \u00a0signalling\t\r \u00a0to\t\r \u00a0actin\t\r \u00a0cytoskeletal\t\r \u00a0dynamics.\t\r \u00a0 Nat\t\r \u00a0Cell\t\r \u00a0Biol\t\r \u00a01,\t\r \u00a0253-\u00ad\u2010259\t\r \u00a0(1999).\t\r \u00a0524.\t\r \u00a0 S.\t\r \u00a0Hino,\t\r \u00a0C.\t\r \u00a0Tanji,\t\r \u00a0K.\t\r \u00a0I.\t\r \u00a0Nakayama,\t\r \u00a0A.\t\r \u00a0Kikuchi,\t\r \u00a0Phosphorylation\t\r \u00a0of\t\r \u00a0beta-\u00ad\u2010catenin\t\r \u00a0by\t\r \u00a0cyclic\t\r \u00a0AMP-\u00ad\u2010dependent\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a0stabilizes\t\r \u00a0beta-\u00ad\u2010catenin\t\r \u00a0through\t\r \u00a0inhibition\t\r \u00a0of\t\r \u00a0its\t\r \u00a0ubiquitination.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Biol\t\r \u00a025,\t\r \u00a09063-\u00ad\u20109072\t\r \u00a0(2005).\t\r \u00a0525.\t\r \u00a0 M.\t\r \u00a0Tomas-\u00ad\u2010Zuber,\t\r \u00a0J.\t\r \u00a0L.\t\r \u00a0Mary,\t\r \u00a0W.\t\r \u00a0Lesslauer,\t\r \u00a0Control\t\r \u00a0sites\t\r \u00a0of\t\r \u00a0ribosomal\t\r \u00a0S6\t\r \u00a0kinase\t\r \u00a0B\t\r \u00a0and\t\r \u00a0persistent\t\r \u00a0activation\t\r \u00a0through\t\r \u00a0tumor\t\r \u00a0necrosis\t\r \u00a0factor.\t\r \u00a0J\t\r \u00a0Biol\t\r \u00a0Chem\t\r \u00a0275,\t\r \u00a023549-\u00ad\u201023558\t\r \u00a0(2000).\t\r \u00a0526.\t\r \u00a0 L.\t\r \u00a0Wang,\t\r \u00a0T.\t\r \u00a0E.\t\r \u00a0Harris,\t\r \u00a0J.\t\r \u00a0C.\t\r \u00a0Lawrence,\t\r \u00a0Jr.,\t\r \u00a0Regulation\t\r \u00a0of\t\r \u00a0proline-\u00ad\u2010rich\t\r \u00a0Akt\t\r \u00a0substrate\t\r \u00a0of\t\r \u00a040\t\r \u00a0kDa\t\r \u00a0(PRAS40)\t\r \u00a0function\t\r \u00a0by\t\r \u00a0mammalian\t\r \u00a0target\t\r \u00a0of\t\r \u00a0rapamycin\t\r \u00a0complex\t\r \u00a01\t\r \u00a0(mTORC1)-\u00ad\u2010mediated\t\r \u00a0phosphorylation.\t\r \u00a0J\t\r \u00a0Biol\t\r \u00a0Chem\t\r \u00a0283,\t\r \u00a015619-\u00ad\u201015627\t\r \u00a0(2008).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 182\t\r \u00a0 527.\t\r \u00a0 T.\t\r \u00a0Kwon,\t\r \u00a0D.\t\r \u00a0Y.\t\r \u00a0Kwon,\t\r \u00a0J.\t\r \u00a0Chun,\t\r \u00a0J.\t\r \u00a0H.\t\r \u00a0Kim,\t\r \u00a0S.\t\r \u00a0S.\t\r \u00a0Kang,\t\r \u00a0Akt\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a0inhibits\t\r \u00a0Rac1-\u00ad\u2010GTP\t\r \u00a0binding\t\r \u00a0through\t\r \u00a0phosphorylation\t\r \u00a0at\t\r \u00a0serine\t\r \u00a071\t\r \u00a0of\t\r \u00a0Rac1.\t\r \u00a0J\t\r \u00a0Biol\t\r \u00a0Chem\t\r \u00a0 275,\t\r \u00a0423-\u00ad\u2010428\t\r \u00a0(2000).\t\r \u00a0528.\t\r \u00a0 R.\t\r \u00a0E.\t\r \u00a0Wettenhall,\t\r \u00a0E.\t\r \u00a0Erikson,\t\r \u00a0J.\t\r \u00a0L.\t\r \u00a0Maller,\t\r \u00a0Ordered\t\r \u00a0multisite\t\r \u00a0phosphorylation\t\r \u00a0of\t\r \u00a0Xenopus\t\r \u00a0ribosomal\t\r \u00a0protein\t\r \u00a0S6\t\r \u00a0by\t\r \u00a0S6\t\r \u00a0kinase\t\r \u00a0II.\t\r \u00a0J\t\r \u00a0Biol\t\r \u00a0Chem\t\r \u00a0267,\t\r \u00a09021-\u00ad\u20109027\t\r \u00a0(1992).\t\r \u00a0529.\t\r \u00a0 A.\t\r \u00a0Moritz,\t\r \u00a0Y.\t\r \u00a0Li,\t\r \u00a0A.\t\r \u00a0Guo,\t\r \u00a0J.\t\r \u00a0Villen,\t\r \u00a0Y.\t\r \u00a0Wang,\t\r \u00a0J.\t\r \u00a0MacNeill,\t\r \u00a0J.\t\r \u00a0Kornhauser,\t\r \u00a0K.\t\r \u00a0Sprott,\t\r \u00a0J.\t\r \u00a0Zhou,\t\r \u00a0A.\t\r \u00a0Possemato,\t\r \u00a0J.\t\r \u00a0M.\t\r \u00a0Ren,\t\r \u00a0P.\t\r \u00a0Hornbeck,\t\r \u00a0L.\t\r \u00a0C.\t\r \u00a0Cantley,\t\r \u00a0S.\t\r \u00a0P.\t\r \u00a0Gygi,\t\r \u00a0J.\t\r \u00a0Rush,\t\r \u00a0M.\t\r \u00a0J.\t\r \u00a0Comb,\t\r \u00a0Akt-\u00ad\u2010RSK-\u00ad\u2010S6\t\r \u00a0kinase\t\r \u00a0signaling\t\r \u00a0networks\t\r \u00a0activated\t\r \u00a0by\t\r \u00a0oncogenic\t\r \u00a0receptor\t\r \u00a0tyrosine\t\r \u00a0kinases.\t\r \u00a0Sci\t\r \u00a0Signal\t\r \u00a03,\t\r \u00a0ra64\t\r \u00a0(2010).\t\r \u00a0530.\t\r \u00a0 K.\t\r \u00a0N.\t\r \u00a0Dalby,\t\r \u00a0N.\t\r \u00a0Morrice,\t\r \u00a0F.\t\r \u00a0B.\t\r \u00a0Caudwell,\t\r \u00a0J.\t\r \u00a0Avruch,\t\r \u00a0P.\t\r \u00a0Cohen,\t\r \u00a0Identification\t\r \u00a0of\t\r \u00a0regulatory\t\r \u00a0phosphorylation\t\r \u00a0sites\t\r \u00a0in\t\r \u00a0mitogen-\u00ad\u2010activated\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a0(MAPK)-\u00ad\u2010activated\t\r \u00a0protein\t\r \u00a0kinase-\u00ad\u20101a\/p90rsk\t\r \u00a0that\t\r \u00a0are\t\r \u00a0inducible\t\r \u00a0by\t\r \u00a0MAPK.\t\r \u00a0J\t\r \u00a0 Biol\t\r \u00a0Chem\t\r \u00a0273,\t\r \u00a01496-\u00ad\u20101505\t\r \u00a0(1998).\t\r \u00a0531.\t\r \u00a0 A.\t\r \u00a0Faisal,\t\r \u00a0M.\t\r \u00a0el-\u00ad\u2010Shemerly,\t\r \u00a0D.\t\r \u00a0Hess,\t\r \u00a0Y.\t\r \u00a0Nagamine,\t\r \u00a0Serine\/threonine\t\r \u00a0phosphorylation\t\r \u00a0of\t\r \u00a0ShcA.\t\r \u00a0Regulation\t\r \u00a0of\t\r \u00a0protein-\u00ad\u2010tyrosine\t\r \u00a0phosphatase-\u00ad\u2010pest\t\r \u00a0binding\t\r \u00a0and\t\r \u00a0involvement\t\r \u00a0in\t\r \u00a0insulin\t\r \u00a0signaling.\t\r \u00a0J\t\r \u00a0Biol\t\r \u00a0Chem\t\r \u00a0277,\t\r \u00a030144-\u00ad\u201030152\t\r \u00a0(2002).\t\r \u00a0532.\t\r \u00a0 T.\t\r \u00a0E.\t\r \u00a0Kmiecik,\t\r \u00a0D.\t\r \u00a0Shalloway,\t\r \u00a0Activation\t\r \u00a0and\t\r \u00a0suppression\t\r \u00a0of\t\r \u00a0pp60c-\u00ad\u2010src\t\r \u00a0transforming\t\r \u00a0ability\t\r \u00a0by\t\r \u00a0mutation\t\r \u00a0of\t\r \u00a0its\t\r \u00a0primary\t\r \u00a0sites\t\r \u00a0of\t\r \u00a0tyrosine\t\r \u00a0phosphorylation.\t\r \u00a0Cell\t\r \u00a049,\t\r \u00a065-\u00ad\u201073\t\r \u00a0(1987).\t\r \u00a0533.\t\r \u00a0 Y.\t\r \u00a0Keshet,\t\r \u00a0R.\t\r \u00a0Seger,\t\r \u00a0The\t\r \u00a0MAP\t\r \u00a0kinase\t\r \u00a0signaling\t\r \u00a0cascades:\t\r \u00a0a\t\r \u00a0system\t\r \u00a0of\t\r \u00a0hundreds\t\r \u00a0of\t\r \u00a0components\t\r \u00a0regulates\t\r \u00a0a\t\r \u00a0diverse\t\r \u00a0array\t\r \u00a0of\t\r \u00a0physiological\t\r \u00a0functions.\t\r \u00a0Methods\t\r \u00a0 Mol\t\r \u00a0Biol\t\r \u00a0661,\t\r \u00a03-\u00ad\u201038\t\r \u00a0(2010).\t\r \u00a0534.\t\r \u00a0 Y.\t\r \u00a0Obara,\t\r \u00a0N.\t\r \u00a0Nakahata,\t\r \u00a0The\t\r \u00a0signaling\t\r \u00a0pathway\t\r \u00a0leading\t\r \u00a0to\t\r \u00a0extracellular\t\r \u00a0signal-\u00ad\u2010regulated\t\r \u00a0kinase\t\r \u00a05\t\r \u00a0(ERK5)\t\r \u00a0activation\t\r \u00a0via\t\r \u00a0G-\u00ad\u2010proteins\t\r \u00a0and\t\r \u00a0ERK5-\u00ad\u2010dependent\t\r \u00a0neurotrophic\t\r \u00a0effects.\t\r \u00a0Mol\t\r \u00a0Pharmacol\t\r \u00a077,\t\r \u00a010-\u00ad\u201016\t\r \u00a0(2010).\t\r \u00a0535.\t\r \u00a0 N.\t\r \u00a0Mody,\t\r \u00a0D.\t\r \u00a0G.\t\r \u00a0Campbell,\t\r \u00a0N.\t\r \u00a0Morrice,\t\r \u00a0M.\t\r \u00a0Peggie,\t\r \u00a0P.\t\r \u00a0Cohen,\t\r \u00a0An\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0the\t\r \u00a0phosphorylation\t\r \u00a0and\t\r \u00a0activation\t\r \u00a0of\t\r \u00a0extracellular-\u00ad\u2010signal-\u00ad\u2010regulated\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a05\t\r \u00a0(ERK5)\t\r \u00a0by\t\r \u00a0mitogen-\u00ad\u2010activated\t\r \u00a0protein\t\r \u00a0kinase\t\r \u00a0kinase\t\r \u00a05\t\r \u00a0(MKK5)\t\r \u00a0in\t\r \u00a0vitro.\t\r \u00a0Biochem\t\r \u00a0J\t\r \u00a0372,\t\r \u00a0567-\u00ad\u2010575\t\r \u00a0(2003).\t\r \u00a0536.\t\r \u00a0 N.\t\r \u00a0Maekawa,\t\r \u00a0J.\t\r \u00a0Abe,\t\r \u00a0T.\t\r \u00a0Shishido,\t\r \u00a0S.\t\r \u00a0Itoh,\t\r \u00a0B.\t\r \u00a0Ding,\t\r \u00a0V.\t\r \u00a0K.\t\r \u00a0Sharma,\t\r \u00a0S.\t\r \u00a0S.\t\r \u00a0Sheu,\t\r \u00a0B.\t\r \u00a0C.\t\r \u00a0Blaxall,\t\r \u00a0B.\t\r \u00a0C.\t\r \u00a0Berk,\t\r \u00a0Inhibiting\t\r \u00a0p90\t\r \u00a0ribosomal\t\r \u00a0S6\t\r \u00a0kinase\t\r \u00a0prevents\t\r \u00a0(Na+)-\u00ad\u2010H+\t\r \u00a0exchanger-\u00ad\u2010mediated\t\r \u00a0cardiac\t\r \u00a0ischemia-\u00ad\u2010reperfusion\t\r \u00a0injury.\t\r \u00a0Circulation\t\r \u00a0113,\t\r \u00a02516-\u00ad\u20102523\t\r \u00a0(2006).\t\r \u00a0537.\t\r \u00a0 W.\t\r \u00a0Antonin,\t\r \u00a0C.\t\r \u00a0Holroyd,\t\r \u00a0D.\t\r \u00a0Fasshauer,\t\r \u00a0S.\t\r \u00a0Pabst,\t\r \u00a0G.\t\r \u00a0F.\t\r \u00a0Von\t\r \u00a0Mollard,\t\r \u00a0R.\t\r \u00a0Jahn,\t\r \u00a0A\t\r \u00a0SNARE\t\r \u00a0complex\t\r \u00a0mediating\t\r \u00a0fusion\t\r \u00a0of\t\r \u00a0late\t\r \u00a0endosomes\t\r \u00a0defines\t\r \u00a0conserved\t\r \u00a0properties\t\r \u00a0of\t\r \u00a0SNARE\t\r \u00a0structure\t\r \u00a0and\t\r \u00a0function.\t\r \u00a0EMBO\t\r \u00a0J\t\r \u00a019,\t\r \u00a06453-\u00ad\u20106464\t\r \u00a0(2000).\t\r \u00a0538.\t\r \u00a0 A.\t\r \u00a0Achuthan,\t\r \u00a0P.\t\r \u00a0Masendycz,\t\r \u00a0J.\t\r \u00a0A.\t\r \u00a0Lopez,\t\r \u00a0T.\t\r \u00a0Nguyen,\t\r \u00a0D.\t\r \u00a0E.\t\r \u00a0James,\t\r \u00a0M.\t\r \u00a0J.\t\r \u00a0Sweet,\t\r \u00a0J.\t\r \u00a0A.\t\r \u00a0Hamilton,\t\r \u00a0G.\t\r \u00a0M.\t\r \u00a0Scholz,\t\r \u00a0Regulation\t\r \u00a0of\t\r \u00a0the\t\r \u00a0endosomal\t\r \u00a0SNARE\t\r \u00a0protein\t\r \u00a0syntaxin\t\r \u00a07\t\r \u00a0by\t\r \u00a0colony-\u00ad\u2010stimulating\t\r \u00a0factor\t\r \u00a01\t\r \u00a0in\t\r \u00a0macrophages.\t\r \u00a0Mol\t\r \u00a0Cell\t\r \u00a0Biol\t\r \u00a028,\t\r \u00a06149-\u00ad\u20106159\t\r \u00a0(2008).\t\r \u00a0539.\t\r \u00a0 M.\t\r \u00a0Pozuelo\t\r \u00a0Rubio,\t\r \u00a0M.\t\r \u00a0Peggie,\t\r \u00a0B.\t\r \u00a0H.\t\r \u00a0Wong,\t\r \u00a0N.\t\r \u00a0Morrice,\t\r \u00a0C.\t\r \u00a0MacKintosh,\t\r \u00a014-\u00ad\u20103-\u00ad\u20103s\t\r \u00a0regulate\t\r \u00a0fructose-\u00ad\u20102,6-\u00ad\u2010bisphosphate\t\r \u00a0levels\t\r \u00a0by\t\r \u00a0binding\t\r \u00a0to\t\r \u00a0PKB-\u00ad\u2010phosphorylated\t\r \u00a0cardiac\t\r \u00a0fructose-\u00ad\u20102,6-\u00ad\u2010bisphosphate\t\r \u00a0kinase\/phosphatase.\t\r \u00a0EMBO\t\r \u00a0J\t\r \u00a022,\t\r \u00a03514-\u00ad\u20103523\t\r \u00a0(2003).\t\r \u00a0 \t\r \u00a0\t\r \u00a0 183\t\r \u00a0 540.\t\r \u00a0 I.\t\r \u00a0Ruvinsky,\t\r \u00a0O.\t\r \u00a0Meyuhas,\t\r \u00a0Ribosomal\t\r \u00a0protein\t\r \u00a0S6\t\r \u00a0phosphorylation:\t\r \u00a0from\t\r \u00a0protein\t\r \u00a0synthesis\t\r \u00a0to\t\r \u00a0cell\t\r \u00a0size.\t\r \u00a0Trends\t\r \u00a0Biochem\t\r \u00a0Sci\t\r \u00a031,\t\r \u00a0342-\u00ad\u2010348\t\r \u00a0(2006).\t\r \u00a0541.\t\r \u00a0 J.\t\r \u00a0E.\t\r \u00a0Galan,\t\r \u00a0J.\t\r \u00a0Pace,\t\r \u00a0M.\t\r \u00a0J.\t\r \u00a0Hayman,\t\r \u00a0Involvement\t\r \u00a0of\t\r \u00a0the\t\r \u00a0epidermal\t\r \u00a0growth\t\r \u00a0factor\t\r \u00a0receptor\t\r \u00a0in\t\r \u00a0the\t\r \u00a0invasion\t\r \u00a0of\t\r \u00a0cultured\t\r \u00a0mammalian\t\r \u00a0cells\t\r \u00a0by\t\r \u00a0Salmonella\t\r \u00a0typhimurium.\t\r \u00a0Nature\t\r \u00a0357,\t\r \u00a0588-\u00ad\u2010589\t\r \u00a0(1992).\t\r \u00a0542.\t\r \u00a0 A.\t\r \u00a0K.\t\r \u00a0Yocum,\t\r \u00a0A.\t\r \u00a0M.\t\r \u00a0Chinnaiyan,\t\r \u00a0Current\t\r \u00a0affairs\t\r \u00a0in\t\r \u00a0quantitative\t\r \u00a0targeted\t\r \u00a0proteomics:\t\r \u00a0multiple\t\r \u00a0reaction\t\r \u00a0monitoring-\u00ad\u2010mass\t\r \u00a0spectrometry.\t\r \u00a0Brief\t\r \u00a0Funct\t\r \u00a0 Genomic\t\r \u00a0Proteomic\t\r \u00a08,\t\r \u00a0145-\u00ad\u2010157\t\r \u00a0(2009).\t\r \u00a0\t\r \u00a0\t\r \u00a0 \t\r \u00a0\t\r \u00a0 184\t\r \u00a0 Appendix\t\r \u00a0 Appendix\t\r \u00a0A\t\r \u00a0Rogers\t\r \u00a0L.D.,\t\r \u00a0Fang\t\r \u00a0Y.,\t\r \u00a0and\t\r \u00a0Foster,\t\r \u00a0L.J.\t\r \u00a0An\t\r \u00a0integrated\t\r \u00a0global\t\r \u00a0strategy\t\r \u00a0for\t\r \u00a0cell\t\r \u00a0lysis,\t\r \u00a0 fractionation,\t\r \u00a0enrichment\t\r \u00a0and\t\r \u00a0mass\t\r \u00a0spectrometric\t\r \u00a0analysis\t\r \u00a0of\t\r \u00a0phosphorylated\t\r \u00a0 peptides.\t\r \u00a0Molecular\t\r \u00a0BioSystems.\t\r \u00a0(2010).\t\r \u00a0 \t\r \u00a0Rogers,\t\r \u00a0L.D.\t\r \u00a0and\t\r \u00a0Foster,\t\r \u00a0L.J.\t\r \u00a0Phosphoproteomics\t\r \u00a0\u2013\t\r \u00a0finally\t\r \u00a0fulfilling\t\r \u00a0the\t\r \u00a0promise?\t\r \u00a0 Molecular\t\r \u00a0BioSystems.\t\r \u00a0(2009).\t\r \u00a0\t\r \u00a0Rogers,\t\r \u00a0L.D.\t\r \u00a0and\t\r \u00a0Foster,\t\r \u00a0L.J.\t\r \u00a0Contributions\t\r \u00a0of\t\r \u00a0proteomics\t\r \u00a0to\t\r \u00a0understanding\t\r \u00a0 phagosome\t\r \u00a0maturation.\t\r \u00a0Cellular\t\r \u00a0Microbiology.\t\r \u00a0(2008).\t\r \u00a0\t\r \u00a0Rogers,\t\r \u00a0L.D.,\t\r \u00a0Kristensen,\t\r \u00a0A.R.,\t\r \u00a0Boyle,\t\r \u00a0E.C.,\t\r \u00a0Robinson,\t\r \u00a0D.P.,\t\r \u00a0Ly,\t\r \u00a0R.T.,\t\r \u00a0Finlay,\t\r \u00a0B.B.\t\r \u00a0and\t\r \u00a0Foster,\t\r \u00a0L.J.\t\r \u00a0Identification\t\r \u00a0of\t\r \u00a0cognate\t\r \u00a0host\t\r \u00a0targets\t\r \u00a0and\t\r \u00a0specific\t\r \u00a0ubiquitylation\t\r \u00a0 sites\t\r \u00a0on\t\r \u00a0the\t\r \u00a0Salmonella\t\r \u00a0SPI-\u00ad\u20101\t\r \u00a0effector\t\r \u00a0SopB\/SigD.\t\r \u00a0Journal\t\r \u00a0of\t\r \u00a0Proteomics.\t\r \u00a0(2008).\t\r \u00a0\t\r \u00a0Rogers,\t\r \u00a0L.D.\t\r \u00a0and\t\r \u00a0Foster,\t\r \u00a0L.J.\t\r \u00a0The\t\r \u00a0dynamic\t\r \u00a0phagosomal\t\r \u00a0proteome\t\r \u00a0and\t\r \u00a0the\t\r \u00a0 contribution\t\r \u00a0of\t\r \u00a0the\t\r \u00a0endoplasmic\t\r \u00a0reticulum.\t\r \u00a0Proceedings\t\r \u00a0of\t\r \u00a0the\t\r \u00a0National\t\r \u00a0Academy\t\r \u00a0 of\t\r \u00a0Sciences.\t\r \u00a0(2007).\t\r \u00a0\t\r \u00a0 Phosphoproteomics\u2014finally fulfilling the promise? Lindsay D. Rogers and Leonard J. Foster* Received 20th March 2009, Accepted 28th May 2009 First published as an Advance Article on the web 7th July 2009 DOI: 10.1039\/b905580k Networks of protein\u2013protein and protein\u2013metabolite interactions are commonly found in biological systems where signals must be passed from one location or component within a cell to another, such as from a receptor on the plasma membrane to a transcription factor in the nucleus. Regulation of such networks, or signal transduction pathways, is often achieved by transient, reversible modification of the components involved. Several types of post-translational modifications of proteins are employed in signal transduction including ubiquitylation of lysines and palmitoylation of cysteines, but by far the best appreciated and apparently the most important involves phosphorylation of serine, threonine and tyrosine residues. Whilst protein phosphorylation has long been recognized as functionally important, low stoichiometry has ultimately impeded global analyses (phosphoproteomics). Recent developments in the application of metal oxide chromatography and advanced mass spectrometric techniques have enabled phosphoproteomics to move beyond mere proof-of-principle experiments, to the stage where it can successfully address complex biological questions. Here we cover the development of phosphopeptide\/protein analysis by mass spectrometry and the various techniques used to enrich phosphopeptides\/proteins. We also speculate on the future of phosphoproteomic research, now that the goal of generating global phosphoproteomic datasets has been realized. History and current potential Signal transduction pathways have classically been studied with a certain degree of myopia; studies have focused on one or a few components out of necessity since no tools were available to allow more global approaches. As more and more targeted tools (e.g., phospho-specific antibodies, specific point mutations) were developed it became clear that there is a great deal of cross-talk and redundancy in signalling pathways, but still the complexity of these systems was generally not fully appreciated. Initially Edman1 degradation was the method of choice for identifying phosphorylation sites in proteins. However, although the process can be automated,2 as a method it suffers from low sensitivity (i.e., 10 to 100 pmol starting material required) and low throughput (i.e., hours to days for one peptide). With the advent of soft ionization methods, protein and peptide mass spectrometry slowly became the method of choice for identifying phosphorylation sites, but for many years one still needed several picomoles of a purified protein to have a reasonable chance of identifying phosphorylation sites that are usually sub-stoichiometric. Thus, while one could identify one or a few sites, phosphorylation analysis on a proteome-scale (i.e., phosphoproteomics) was not possible. Two-dimensional gel electrophoresis, initially using autoradiography to detect 32P-labelled proteins and then using colorimetric or fluorimetric imaging and phosphate-specific dyes, allowed the visualization of phosphoproteomes, but determining the specific sites of phosphorylation remained an insensitive and slow process. The Department of Biochemistry & Molecular Biology, Centre for High-Throughput Biology, University of British Columbia, Vancouver, BC, Canada. E-mail: ljfoster@interchange.ubc.ca Lindsay D. Rogers Lindsay D. Rogers is a doctoral candidate in Biochemistry and Molecular Biology at the University of British Columbia. A graduate of Queens University, she is studying how Salmonella bacteria subvert host cell processes. Leonard J. Foster Leonard J. Foster is an Assis- tant Professor of Biochemistry and Molecular Biology at the University of British Columbia in Vancouver, Canada. He is a founding member of the Centre for High-Throughput Biology and is interested in the molecular events underlying host-pathogen interactions. 1122 | Mol. BioSyst., 2009, 5, 1122\u20131129 This journal is \u0002c The Royal Society of Chemistry 2009 REVIEW www.rsc.org\/molecularbiosystems | Molecular BioSystems D ow nl oa de d by T he U ni ve rs ity o f B rit ish C ol um bi a Li br ar y on 1 2 A pr il 20 11 Pu bl ish ed o n 07 Ju ly 2 00 9 on h ttp :\/\/ pu bs .rs c.o rg | d oi: 10. 103 9\/B 905 580 K View Online effective coupling of nano or capillary-flow high performance liquid chromatography (HPLC) to tandem mass spectrometry (MSn) by electrospray ionization (ESI) has made possible the analysis of highly complex and dynamic proteomes. The structural information provided by information rich MSn experiments3 also provides a suitable platform for characterization of phosphoproteins. However, five apparent problems were facing this approach: lability of the phosphate modification, the anionic nature of the phosphate, ion suppression, lack of retention of phosphopeptides on reversed phase stationary phases and phosphorylation stoichiometry. We use the term \u2018apparent problems\u2019 because evidence now suggests that the lability of the phosphate moiety and its anionic nature, as well as its retention on reversed phase, are not particularly problematic. It is also evident that poor phosphopeptide detection is likely more a problem of low stoichiometry between the phosphorylated and non- phosphorylated form of proteins (inducing ion suppression during liquid chromatography\u2013tandem mass spectrometry (LC-MS\/MS)), as opposed to a reduced ionization efficiency of phosphopeptides per se. While stoichiometry presents what is likely the most significant challenge, we aim to address this as well as the lability of the phosphate modification in subsequent sections. With regard to the difficulties of working with a modification that has a reasonable chance of holding a negative charge even under very acidic conditions (e.g., pH 2.0), this too has proven to be less of an impediment than previously thought. Certainly it makes some chemical sense that conventional LC-MS\/MS of tryptic peptides at low pH and in positive ion mode was incompatible with phosphorylation analysis; the positive charges on the peptide due to amine protonation would be at least partially cancelled out by the negative charge on the phosphate. It was expected that this would tend to leave only singly-charged peptides, which are not optimal for MS\/MS. It may be that preparing phosphopeptide samples in strong acids such as trifluoroacetic acid achieves sufficient acidification of phosphorylated peptides, as the authors have observed no increase in the number of phosphopeptides identified, nor the distribution of the number of phosphorylation sites per peptide by including singly-charged ions for MS\/MS (unpublished data). With regard to phosphorylated peptides not being retained on C18 reversed phase, the favoured stationary phase for LC-MS\/MS analysis of peptides, evidence clearly indicates that the methods used for resolving unphosphorylated peptides are perfectly amenable to phosphopeptides as well. Indeed, if anything phosphopeptides are likely slightly better retained by C18 than their unphosphorylated counterparts,4 although two other studies found essentially no difference in retention times.5,6 However, running reversed phase LC-MS\/MS under lower pH conditions (pH 1.7, 3% formic acid) has been observed to improve the detection of poly- phosphorylated peptides.7 The total ion chromatogram shown in Fig. 1 compares the elution times of all peptides identified from a complex sample (tryptic digest of mammalian cytosolic fraction) enriched for phosphopeptides. Consistent with previous reports, the elution patterns of phosphopeptides and non-phosphopeptides are very similar. With regard to poor phosphopeptide detection, for many years it was believed that phosphopeptides are not effectively detected because of their poor ionization efficiency. There is still some controversy surrounding this point, with one study suggesting that phosphopeptides ionize more or less as efficiently as their unphosphorylated counterparts.6 A more recent study found that by treating phosphopeptide-enriched samples with alkaline phosphatase, the intensities of the dephosphorylated forms of peptides were, on average, two-fold higher than the intensities of the phosphorylated species.5 The former study has been criticized for analysing primarily non-tryptic peptides when virtually everyone in the field uses trypsin to digest proteins. On the other hand, the latter study did not take into account the likely sub-stoichiometric levels of phosphorylation in their samples. This would have overlooked the additive effect of having some unphosphorylated peptide signal contributing to the signal from the newly-dephosphorylated peptides. In any case, in the context of a complex mixture where phosphopeptides are present only at their natural stoichiometry, they would appear to ionize less efficiently but the problem in such a case is the stoichiometry and thus the dynamic range of the instrumentation rather than the ionizability. Lysis methods and low stoichiometry of phosphorylated proteins An ongoing challenge in phosphoproteomics is the low phosphorylation signal typically observed in biological samples. Due to the reversible nature of protein phosphorylation, phosphatases liberated upon cell lysis can quickly and substantially reduce the signal. In addition, large amounts of starting material (typically milligrams) and extremely sensitive instrumentation are required, especially as protein phos- phorylation emerges as an important, yet rare, modification in prokaryotes.8\u201310 While a similar number of kinases and phosphates are thought to regulate tyrosine phosphorylation, many more serine\/threonine kinases exist than cognate phosphatases in Fig. 1 Comparing elution times of phosphorylated and non- phosphorylated peptides from a C18 reversed phase column. The total ion chromatogram is shown, with the elution time versus mass-to-charge ratio of all peptides detected in the sample overlaid over top (phosphopeptides in red, non-phosphopeptides in blue). This journal is \u0002c The Royal Society of Chemistry 2009 Mol. BioSyst., 2009, 5, 1122\u20131129 | 1123 D ow nl oa de d by T he U ni ve rs ity o f B rit ish C ol um bi a Li br ar y on 1 2 A pr il 20 11 Pu bl ish ed o n 07 Ju ly 2 00 9 on h ttp :\/\/ pu bs .rs c.o rg | d oi: 10. 103 9\/B 905 580 K View Online the human genome.11 Also, kinase-substrate specificity is typically determined by the amino acid sequence surrounding the phosphorylated residue, while phosphatases are thought to rely on targeting subunits to achieve specificity.12 This has made phosphatases difficult to study and our understanding of them lags considerably that of kinases. By far the most common means of inhibiting phosphatases in phosphoproteomic studies is through the use of commercially available inhibitors. While vanadium oxides such as pervanadate and orthovanadate are used to inhibit all protein tyrosine phosphatases (PTPs), about 20 families of serine\/threonine phosphatases have been classified, the most common of which are protein phosphatase 1 (PP1), PP2A and PP2B. Phosphatase inhibitors such as calyculin A are commonly used to inhibit PP1 and PP2A, while inhibitors such as deltamethrin have been shown to inhibit PP2B. However, in a recent study Pan et al. treated live Hepa1-6 liver cells with pervanadate, calyculin A, and deltamethrin and compared the abundance of individual phosphorylated peptides to an untreated condition.9 Surprisingly, only 27% of peptides were found to increase more than two-fold following this treatment, suggesting that either the phosphatases targeting many of the sites are not affected by the inhibitors, the dephosphorylation of those sites have absurdly slow kinetics, or almost three quarters of phos- phorylations are near stoichiometric in growing cells (which is not the case). Interestingly, Pan et al. also found that the majority of pY sites were up-regulated more than two-fold (70%), presumably due to the broad inhibition of PTPs by pervanadate and perhaps also the low level of tyrosine phos- phorylation in untreated cells. Only 41% of phosphothreonine and 26% of phosphoserine sites were up-regulated two-fold during treatment with these widely used inhibitors, which are believed to block most phosphatase activity. Similarly, in an analysis of the stem cell plasma membrane phosphoproteome, Thingholm et al. found that pre-treating cells with calyculin A, sodium pervanadate, or two phosphatase inhibitor cocktails resulted in only a 10\u201340% increase in the number of phosphoproteins identified.13 Again, inhibition with calyculin A resulted in an 88.5% increase in the number of phospho- tyrosine containing proteins identified. Thus, while current inhibitor-based methods of inactivating phosphatases are somewhat effective, especially when considering tyrosine phosphorylation, for truly global phosphoproteomic analyses alternative methods of phosphatase inactivation, such as heat or chaotropic denaturation should be considered. Phosphorylation of serine, threonine and tyrosine residues has long since been documented in eukaryotes from yeast to humans,14,15 but only recently with the emergence of large-scale phosphoproteomics studies has it also emerged as an important and widespread regulatory mechanism in prokaryotes. Reporting the first large-scale site-specific phospho- proteomics study in bacteria, Macek et al. identified 103 unique phosphopeptides from 78 Bacillus subtilis proteins, also observing that the distribution of phosphoserine, -threonine and -tyrosine residues (B70 : 20 : 10) closely resembles that of multicellular organisms.16 They also observed phosphoproteins in all parts of the bacterial cell and among a wide variety of metabolic and regulatory enzymes, especially those involved in carbohydrate metabolism. The extent of serine, threonine and tyrosine phosphorylation in B. subtilis was found to be at least an order of magnitude less than that of mammalian cells, phosphoproteins were found to be overrepresented in essential genes, and the bacterial phosphorylation sites identified do not match common target sequences for eukaryotic kinases which suggests that many of them have evolved independently in bacteria. In a follow-up study, similar results were obtained in Escherichia coli, again finding no similarity to eukaryotic kinase motifs and also limited similarity between phosphorylation sites in B. subtilis and E. coli (20%).17 Interestingly, about twice the number of phosphorylated proteins in B. subtilis and E. coli are conserved among other phylogenetically distant organisms as compared to non- phosphorylated proteins, and nine (B10%) were found to be conserved from archaea to humans. Additional studies on Campylobacter jejuni and Lactococcus lactus yielded similar results, solidifying the emergence of bacterial phosphorylation on serine, threonine, and tyrosine residues as a significant regulatory mechanism in both prokaryotic and eukaryotic organisms.18,19 The need for pre-fractionation and enrichment Although it is estimated that one third of all mammalian proteins are phosphorylated at some point, only a subset are modified by any given stimulus.20 Furthermore, due to the tight spatial and temporal control observed in signalling pathways, protein phosphorylation events typically occur at very low stoichiometry. Thus, for large-scale phosphoproteomic analyses, enrichment of phosphorylated proteins and peptides is indispensable and several such techniques have been developed. When analysing their results from a large-scale phospho- proteomic analysis of HeLa cells stimulated with epidermal growth factor, Olsen et al. reported that almost half of the phosphorylation events they observed occurred on nuclear proteins, despite the fact that only one third of all proteins in the database were assigned as nuclear by gene ontology.8 Alternatively mitochondrial and plasma membrane proteins were underrepresented, suggesting that subcellular fractionation is required for effective phosphoproteome analysis of individual organelles, as well as for generally improving sensitivity in whole cell phosphoproteomic analyses.8 In addition, several chromatographic methods and, to a lesser extent, calcium and barium precipitation and isoelectric focusing (IEF) have also been used to enrich phosphorylated peptides. At pH 2.7, only lysine, arginine, histidine, and the amino terminus of a peptide are charged and the majority of peptides carry a charge of 2+.21 A negatively charged phosphate moiety reduces this charge state and thus, with increasing salt concentration, phosphopeptides elute earlier than the majority of non-phosphorylated peptides from a strong cation- exchange (SCX) column.10,21,22 In a study of yeast pheromone- signalling, Gruhler et al. utilized an ammonium formate gradient from 5 to 600 mM and retained 40% of the eluate for analysis.22 The use of strong anion-exchange chromatography (SAX) has also been described.23,24 Dai et al. have coupled SAX online to LC-MS\/MS, eluting 1124 | Mol. BioSyst., 2009, 5, 1122\u20131129 This journal is \u0002c The Royal Society of Chemistry 2009 D ow nl oa de d by T he U ni ve rs ity o f B rit ish C ol um bi a Li br ar y on 1 2 A pr il 20 11 Pu bl ish ed o n 07 Ju ly 2 00 9 on h ttp :\/\/ pu bs .rs c.o rg | d oi: 10. 103 9\/B 905 580 K View Online phosphopeptides using a decreasing pH gradient onto the reversed phase column for LC-MS\/MS.25 However, acidic peptides also bind strongly to the material and it has been reported that phosphopeptides bind with very strong affinity and can be difficult to remove from the resin.26 During hydrophilic interaction chromatography (HILIC), peptides are applied to a hydrophilic stationary phase in an organic mobile phase and separation is achieved based on polarity using an inverse gradient from high to low organic content.27,28 Similarly, electrostatic repulsion hydrophilic interaction chromatography (ERLIC) employs HILIC on a weak anion-exchange column (WAX).29 During ERLIC, peptides are run at low pH (B2) in a mobile phase containing high organic, and eluted with increasing salt concentration. Contrary to HILIC, which strongly retains very acidic and basic peptides, and WAX chromatography, which elutes basic peptides very early, ERLIC retains basic peptides due to their hydrophilic interaction and acidic peptides based on their electrostatic interaction to the WAX. With increasing salt concentration phosphopeptides elute last from an ERLIC column with monophosphorylated peptides eluting earlier than polyphosphorylated peptides. In a study by Gan et al. ERLIC was compared to SCX and demonstrated to yield three times the number of phosphopeptide identifications.30 In addition to these chromatographic methods, Zhang et al. have reported precipitation of phosphopeptides by co-precipitation with calcium phosphate, while Ruse et al. have demonstrated phosphopeptide precipitation in Ba2+ and acetone.31,32 Maccarrone et al. have shown that the acidic nature of phosphopeptides also enables their efficient separation by IEF, which results in the vast majority of phosphopeptides migrating within a pH range of 3 to 6.33 Methyl-esterification of carboxyl groups has been applied to improve separation of phosphopeptides from acidic peptides which otherwise co-migrate to acidic regions during focusing.34,35 However, while traditional IEF provides efficient pre-fractionation of phosphopeptides, more modern systems that do not rely on immobilized pH gradient strips retain the entire sample volume and thus prevent the loss of phospho- peptides that would otherwise migrate off the strip (even at the lowest pH ranges available). Pre-fractionation by in solution IEF separates phosphopeptides from a full cell lysate quite uniformly within a pH range ofB3\u20136 and offers an advantage over both traditional IPG-based IEF and chromatographic methods by minimizing sample loss (unpublished data). The methods described above offer crude phosphopeptide purification, and in some cases effective fractionation of phosphopeptides themselves. However, due to their very low abundance, additional techniques offering very high selectivity for phosphate are often preferred or used in combination and downstream of these methods. For highly selective enrichment of tyrosine phosphorylated peptides and proteins, immunoprecipitation using phospho- tyrosine specific antibodies has proven to be a very efficient technique.36\u201341 In these studies the antibody and bead amounts are optimized for a given system to minimize non- specific binding and to ensure complete immunoprecipitation. However, with the exception of work done by Matsuoka et al. to identify ATM and ATR substrates, poor specificity has been observed for phosphoserine and phosphothreonine antibodies.42 This, combined with the fact that only B2% of eukaryotic phosphorylation sites have been estimated to occur on tyrosine residues, suggests that additional purification techniques are required for truly global analyses of phos- phorylation signalling.8 Immobilized metal affinity chromatography (IMAC) and metal oxide chromatography (MOC) represent two widely used methods for the selective enrichment of phosphoserine, -threonine and -tyrosine containing peptides. IMAC is based on the high affinity of phosphate groups for metal ions such as Fe3+, Zn2+, and Ga3+ 43,44 (Fig. 2a). One of the main limitations of IMAC involves the non-specific retention of generally acidic peptides due to the affinity of negatively charged carboxylates for the positively charged metal ions in the matrix (Fig. 2c). Derivatizing carboxylic acid moieties to methyl esters has been reported to greatly reduce contamination by acidic peptides. However sample handling and\/or reaction conditions used often facilitate the partial deamidation of asparagine and glutamine residues and the resulting aspartate and glutamate residues are then susceptible to methylation, which adds a significant layer of complexity to the analysis. As with any chemical derivatization, methyl-esterification also decreases sensitivity due to sample loss.45\u201348 Alternatively, MOC utilizes the affinity of acidified phosphoric acid for metal oxides such as TiO2 and ZrO2 49 (Fig. 2a). Without requiring chemical modification of carboxylates, MOC makes use of dihydroxy-benzoic acid (DHB), or aliphatic hydroxyl acids (i.e., lactic or b-hydroxypropanoic acid) to compete with acidic but not phosphorylated peptides from the matrix (Fig. 2b) and has been reported to yield a far higher selectivity towards phosphorylated peptides versus carboxylates than does IMAC.50\u201353 Recently, Thingholm et al. have developed a combined approach termed \u2018sequential elution from IMAC\u2019 (SIMAC), which first elutes monophosphorylated and acidic peptides from an IMAC column under acidic conditions, followed by the elution of polyphosphorylated peptides under basic conditions.54 The monophosphorylated peptides are separated from acidic peptides using TiO2 chromatography, the two eluates are analysed separately by LC-MS\/MS, and SIMAC has been reported to more than double the number of phosphopeptides identified by TiO2 chromatography alone. As mentioned, many large-scale phosphoproteomics studies currently use a combination of one of the described crude enrichment steps, followed by either IMAC or MOC to yield near complete selectivity for phosphorylated peptides.8,28,30,55 We have focused primarily on phosphopeptide enrichment techniques that do not require chemical derivatization of the phosphate moiety itself. While several derivatization methods, e.g., b-elimination, have been applied to phosphoproteomic studies and are highly selective, most suffer from significant losses during the derivatization step, which also increases sample complexity through multiple side reactions.56,57 Therefore, we believe that chemical derivatization approaches for enriching phosphopeptides will find limited application in large-scale studies. However, methods such as iTRAQ and SILAC combined with the described highly selective This journal is \u0002c The Royal Society of Chemistry 2009 Mol. BioSyst., 2009, 5, 1122\u20131129 | 1125 D ow nl oa de d by T he U ni ve rs ity o f B rit ish C ol um bi a Li br ar y on 1 2 A pr il 20 11 Pu bl ish ed o n 07 Ju ly 2 00 9 on h ttp :\/\/ pu bs .rs c.o rg | d oi: 10. 103 9\/B 905 580 K View Online enrichment techniques are becoming the gold standard for functional phosphoproteomics research.8,37,40,55 LC-MS\/MS methods for phosphoproteomics The dogma in the mass spectrometry field for several years was that collision-induced dissociation (CID) is ineffective for phosphopeptide analysis as neutral loss of phosphoric acid (H3PO4, from pS and pT) would occur before backbone cleavage, giving insufficient backbone fragmentation for effective identification of the peptide58 (Fig. 3a). Electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD) were recognized to minimize such neutral losses but were, and still are, too slow for HPLC timescales and were restricted to Fourier transform-ion cyclotron resonance (FT-ICR) instruments. Methods such as MS3 and MultiStage Activation have been employed to impose additional activation events on pre-selected neutral loss peaks.21,59 They differ in the sense that during MS3 the peak selected for additional fragmentation is isolated prior to activation and results in an entirely new set of product ions (Fig. 3b), while during MultiStage Activation the second isolation step is eliminated and the spectra contain product ions from both activation events, making it a pseudo-MS3 approach (Fig. 3c). MultiStage Activation also enables higher MSn activations in cases where multiply phosphorylated peptides undergo sequential neutral losses. Following MS3 or MultiStage Activation, phosphoserine and -threonine sites are recognized by the presence of fragment ions containing dehydroalanine and dehydrobutyric acid, respectively, while phosphotyrosine residues are rarely observed to undergo the described neutral loss.60 However, the advantages of using neutral loss-driven MS3 and pseudo-MS3 scans in phosphoproteomics are somewhat controversial. Recently, Villen et al. have shown that MS3-based schemes did not result in an overall increase in the number of phosphopeptides identified and offered only a very minor advantage in phosphosite localization.61 They attribute this to the fact that MS3 based schemes are approximately 20% slower, generally hold only 15% of the ion intensity of MS2 spectra, and rarely produce more informative ions than normalMS2 CIDwhen highmass-accuracy instruments are used. Conversely, Ulintz et al. observed that, using an LTQ-FT, both MultiStage Activation and MS2 outperform MS3 methodologies, MultiStage Activation resulted in a 6% increase in the number of unique phosphopeptides identified, and all three methods performed equally well in localizing phosphorylation sites.62 Overall it appears that while pseudo-MS3 scans may generate slightly richer spectra than MS2 and MS3 scans, mass spectrometers with high mass accuracy and increased ion capacity are capable of producing much richer MS2 scans for phosphoproteomic analyses than was originally realized. In addition, MS3-based scans can complicate analysis and cause ambiguity in phosphosite localization. For example, Kru\u0308ger et al. reported that the neutral loss of methanesulfonic acid from methionine and phosphoric acid from threonine, both yield dehydrobutyric acid. This can impose false positives from MS3 scans where fragment ions containing dehydrobutyric acid are used to localize phosphothreonine sites in instances of methionine and threonine isomerism.63 Similarly, Palumbo et al. have shown that, contrary to the b-elimination reaction that was believed to explain the neutral loss of phosphoric acid from phosphoserine and phosphothreonine-containing peptides, the neutral loss occurs Fig. 2 Diagram of IMAC and TiO2 enrichment methods. (A) The co-ordination of IMAC and TiO2 resin with a phosphorylated peptide is shown. (B) The co-ordination of TiO2 resin with DHB (middle) and lactic acid (right) is shown. (C) The co-ordination of IMAC and TiO2 with a peptide containing an acidic residue (carboxyl group) is shown. Peptides are represented by a red wave line. 1126 | Mol. BioSyst., 2009, 5, 1122\u20131129 This journal is \u0002c The Royal Society of Chemistry 2009 D ow nl oa de d by T he U ni ve rs ity o f B rit ish C ol um bi a Li br ar y on 1 2 A pr il 20 11 Pu bl ish ed o n 07 Ju ly 2 00 9 on h ttp :\/\/ pu bs .rs c.o rg | d oi: 10. 103 9\/B 905 580 K View Online through a SN2 charge-directed mechanism that can result in the sequential neutral loss of metaphosphoric acid and water from two different residues in a peptide.64 In this case, localizing a phosphorylation site based on the presence of dehydroalanine or dehydrobutyric acid may mislocalize the site. Palumbo et al. also observed that during the relatively long activation times required for CID in ion trap mass spectrometers, almost half of the peptides they analyzed had transferred a phosphate group to a previously unmodified site during activation. This was observed to increase with proton mobility and at lower charge states, and also to be dependent on the relatively long activation times employed for CID in an ion trap MS. Thus to minimize such artefacts, peptides should be activated in either a quadrupole, or using alternative fragmentation methods such as electron capture dissociation (ECD) or electron transfer dissociation (ETD), both of which allow fragmentation along the peptide backbone while maintaining the phosphate intact.65,66 Phosphopeptide\/protein databases As the last four years have seen an exponential increase in the number of identified phosphorylation sites, several groups have expended enormous efforts to curate and compile these data into on-line resources. Much of the high quality data, including some high-content data, are curated in UniProt,67 which is probably the best way to make the data available to the wider biological community since it is used outside the high-throughput disciplines. In addition, more specific compendiums are available (Table 1) and each provides specific types of tools (e.g., predictors, network\/pathway viewers) and\/or information (e.g., analytical context in which peptides were identified, quantitative profiles of phosphorylation dynamics after agonist stimulation). The overlap in phospho- peptide information stored in some of these databases is very high so we expect to see many of these efforts combining forces in a few years, as was seen with the protein interaction network databases between 2004 and 2007. Several hurdles face such an effort to reduce redundancy, however, as each database has different standards for data reliability regarding peptide identification and phosphosite localization. Major challenges Here we have attempted to summarize where the field of phosphoproteomics is and how it has developed to this point. Our view of the field is that MOC is very quickly beginning to dominate all other approaches for enriching phosphopeptides. The effectiveness of MOC is enhanced greatly by pre- fractionation approaches and it is not yet clear if one of these approaches is vastly better than the others. In the future, confidence in site localization needs to be better addressed, especially as phenomena such as the transfer of a phosphate during CID and the sequential loss of metaphosphoric acid Fig. 3 Schematic of MS2, MS3 and MultiStage Activation for phosphoproteomics. (A) During MS2 a phosphopeptide is selected for CID and isolated (1A). The resulting fragments consist either of primarily y- and b-ions (2A) or a dominant neutral loss peak corresponding to the loss of phosphoric acid (\u000398 Da) (3A). (B) DuringMS3 a phosphopeptide is selected for CID and isolated (1B). If a dominant neutral loss peak is detected (\u000398 Da) (2B), the neutral loss peak is re-isolated (3B) and an additional round of CID yields fragment ions from the neutral loss peak (4B). (C) During MultiStage Activation a peptide is selected for CID and isolated (1C). If a dominant neutral loss peak is detected (\u000398 Da) (2C) the neutral loss peak is re-activated by CID (2C) yielding a product ion spectra containing fragment ions from both collision events. Red and blue bars represent parent ions and fragment ions from the first and second fragmentation events, respectively. This journal is \u0002c The Royal Society of Chemistry 2009 Mol. BioSyst., 2009, 5, 1122\u20131129 | 1127 D ow nl oa de d by T he U ni ve rs ity o f B rit ish C ol um bi a Li br ar y on 1 2 A pr il 20 11 Pu bl ish ed o n 07 Ju ly 2 00 9 on h ttp :\/\/ pu bs .rs c.o rg | d oi: 10. 103 9\/B 905 580 K View Online and water from different sites are reported. We also hope to see these techniques move out of proof-of-principle studies and begin to be widely applied to address real biological questions, but this will only come by incorporating a quantitative dimension into experiments.8 Metabolic labelling strategies are likely to be favoured over chemical introduction of stable isotopes or label-free approaches for quantitative phosphoproteomics for at least two reasons: (1) chemical derivatization invariably results in some sample loss and the scale required for successful measurement of phosphoproteomes is already big enough, (2) spectral counting approaches are not appropriate in cases where proteins may be identified by only one or two peptides, as in phosphoproteomics. Nonetheless, these technologies exist and are mature enough now to be used for quantitative or functional phosphoproteomics. Many scientists who still practice the \u2018one lab, one protein\u2019 approach to molecular biology take a dim view of \u2018omics approaches\u2019 and until more effective methods for validating a phospho- proteomic dataset become available, such views will persist. Reviewers will still ask proteomics researchers to \u2018validate\u2019 their findings by western blot or other equally out-dated methods, so better tools than antibodies need to be developed for follow-up studies to phosphoproteome analyses. A perfect example is the ease with which one can quickly and effectively screen thousands of different genes through the use of siRNA in Drosophila or Caenorhabditis elegans.74 Since whole genome RNAi screens in mammalian systems remain very challenging, combining the discovery of regulated phos- phorylation sites by quantitative phosphoproteomics with highly-multiplexed multiple reaction monitoring assays for determining stoichiometry and for tracking the levels of sites across a wide range of conditions represents a very attractive goal for the field to strive for in the next five years. Acknowledgements The authors thank the other members of the Cell Biology Proteomics (CBP) group in the Centre for High-Throughput Biology for fruitful discussions and advice, particularly Robert Parker, Anders Kristensen and Nikolay Stoynov. Phosphoproteomic research in the CBP is supported by the Canadian Institutes of Health Research (CIHR), the Canadian Foundation for Innovation, the British Columbia (BC) Knowledge Development Fund and the Michael Smith Foundation through the BC Proteomics Network (BCPN). LJF is the Canada Research Chair in Organelle Proteomics and a Michael Smith Foundation Scholar. LDR is supported by a CIHR PGS-D award. References 1 P. Edman, Acta Chem., Scand., 1950, 4, 283\u2013293. 2 H. D. Niall, Methods Enzymol., 1973, 27, 942\u20131010. 3 R. Aebersold and M. Mann, Nature, 2003, 422, 198\u2013207. 4 Y. Ishihama, F. Y. Wei, K. Aoshima, T. Sato, J. Kuromitsu and Y. Oda, J. Proteome Res., 2007, 6, 1139\u20131144. 5 M. Marcantonio, M. Trost, M. Courcelles, M. Desjardins and P. Thibault, Mol. Cell. Proteomics, 2008, 7, 645\u2013660. 6 H. Steen, J. A. Jebanathirajah, J. Rush, N. Morrice and M. W. Kirschner, Mol. Cell. Proteomics, 2006, 5, 172\u2013181. Table 1 Publicly accessible phosphopeptide databases Name, URL Descriptiona Proteins\/ peptidesb Notesc Ref. Phospho.ELM, http:\/\/phospho.elm.eu.org\/ Experimentally-verified eukaryotic phosphorylation sites 4110\/18 252 2166 pY, 13 320 pS, 2766 pT sites 68 PhosphoPOINT, http:\/\/kinase.bioinformatics.tw\/ Comprehensive human interactome and phosphoprotein database 4195 Integrates phosphoproteins, kinases and their protein\u2013 protein interaction networks 69 Phosida, http:\/\/www.phosida.com\/ Management, structural and evolutionary investigation, and prediction of phosphosites 6518\/15 648 Contains information of temporal regulation of phosphosites by various stimuli 70 PhosPhAt, http:\/\/phosphat.mpimp-golm.mpg.de\/ Arabidopsis thaliana and plant-specific phosphorylation site predictor 6282 peptides Experimental and analytical context information. pSer prediction algorithm 71 P3DB, http:\/\/www.p3db.org\/ Resource of protein phosphorylation data from multiple plants 8554\/11 491 General to all plants 72 PhosphoNET, http:\/\/www.phosphonet.ca\/ Human Phospho-Site Knowledgebase 5374\/26 052 Functional and regulatory information included. Links to UniProt and other phospho databases Corporated PhosphoPep, http:\/\/www.phosphopep.org\/ Project to support systems biology signalling research in model organisms Various Specific foci on Saccharomyces cerevisiae, Drosophila melanogaster, C. elegans, Homo sapiens. Tools for browsing pathways 73 PhosphoSitePlus, http:\/\/www.phosphosite.org\/ Protein modification resource 9888\/64 934 Links to literature and MS\/MS records. Additional modifications included Corporated a The stated purpose of the given database, often taken directly from the on-line information. b The reported number of phosphoproteins and phosphopeptides, where it is explicitly stated. c Unique features of the given database. d Databases developed by corporations, not presented in a peer-reviewed publication. 1128 | Mol. 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Biotechnol., 2008, 26, 1339\u20131340. 74 G. J. Hannon, Nature, 2002, 418, 244\u2013251. This journal is \u0002c The Royal Society of Chemistry 2009 Mol. BioSyst., 2009, 5, 1122\u20131129 | 1129 D ow nl oa de d by T he U ni ve rs ity o f B rit ish C ol um bi a Li br ar y on 1 2 A pr il 20 11 Pu bl ish ed o n 07 Ju ly 2 00 9 on h ttp :\/\/ pu bs .rs c.o rg | d oi: 10. 103 9\/B 905 580 K View Online Microreview Contributions of proteomics to understanding phagosome maturation OnlineOpen: This article is available free online at www.blackwell-synergy.com Lindsay D. Rogers and Leonard J. Foster* Cell Biology Proteomics group, Centre for High-throughput Biology and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada V6T 1Z4. Summary In metazoans macrophage cells use phagocytosis, the process of engulfing large particles, to control the spread of pathogens in the body, to clear dead or dying cells, and to aid in tissue remodelling, while the same process is also used by unicellular eukaryotes to ingest food. Phagocytosing cells essentially swallow the particles, trapping them in vacuoles called phagosomes that go through a series of matu- ration steps, culminating in the destruction of the internalized cargo. Because of their central role in innate immunity and their relatively simple structure (one membrane bilayer surrounding a single particle), phagosomes have been a popular subject for organelle proteomics studies. Qualitative proteomic technologies are now very sensitive so hundreds of different proteins have been identified in phago- somes from several species, revealing new proper- ties of these intriguing compartments. More recently, quantitative proteomic approaches have also been applied, shedding new light on the dynamics and composition of maturing phagosomes. In this review we summarize the studies that have applied pro- teomic technologies to phagosomes and how they have changed our understanding of phagosome biology. Phagocytosis Professional phagocytes of the innate immune system normally destroy invading microorganisms by engulfing them into specialized vacuoles and then conferring micro- bicidal and degradative capabilities upon the new com- partments (Vieira et al., 2002; Underhill, 2005; Haas, 2007). In this process, phagocytic receptors at the cell surface bind to an invading microorganism, inducing receptor clustering, which in turn initiates phagocytosis. Non-opsonic receptors recognize and bind to specific molecules or molecular patterns on the pathogen\u2019s surface, including moieties such as mannyl or fucosyl residues, lipopolysaccharides or lipoproteins. Alterna- tively, host-derived ligands such as complement proteins and immunoglobulins can identify and coat foreign objects and these opsonins are then recognized by cell-based receptors, including the complement receptors and the FcY receptors. Multiple ligands on a single foreign particle force the receptors to cluster, which initiates signalling pathways unique to the receptor\u2013ligand pair. These cas- cades recruit diverse enzymes and structural components that effect extensive membrane remodelling, culminat- ing in the engulfment of the foreign particle within the cell, encased in a membranous organelle called the phagosome. Phagosome maturation Following fission of the phagosome from the plasmale- mma, the classic model has the newly formed organelle fusing sequentially with (maturing into) sorting (or early) endosomes, late endosomes and finally lysosomes to acquire microbicidal and degradative capabilities. Among several proteins delivered by lysosomal fusion, Slc11a1 (Nramp1) appears to be of critical importance in macroph- ages\u2019 ability to effectively deal with bacteria. The mecha- nisms behind this are poorly understood but may involve the regulation of divalent cation concentrations in the phagolysosomal lumen (Forbes and Gros, 2001). Thus, pathogens are not killed by internalization per se, but rather by the extremely hostile lumen of the pha- golysosome. However, the order of the fusion steps is Received 1 February, 2008; revised 4 March, 2008; accepted 5 March, 2008. *For correspondence. E-mail ljfoster@interchange.ubc. ca; Tel. (+1) 604 822 8311; Fax (+1) 604 822 2114. Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation. Cellular Microbiology (2008) 10(7), 1405\u20131412 doi:10.1111\/j.1462-5822.2008.01140.x First published online 26 March 2008 \u00a9 2008 The Authors Journal compilation \u00a9 2008 Blackwell Publishing Ltd highly specific \u2013 newly formed phagosomes only fuse with sorting endosomes, and only after this event can they fuse with late endosomes, and so forth (Vieira et al., 2002). Based on these observations, it is generally agreed that fusion with each compartment delivers the necessary protein machinery to allow subsequent fusion steps to occur. Work in model systems of vesicle fusion has demonstrated roles for soluble N-ethylmaleimide sen- sitive attachement protein receptor (SNARE) proteins and small GTPases of the Rab family in specifying the fusion competence of two membranes and, to this end, several members of these protein families have been shown to localize to one endosome\/phagosome stage or another (Duclos et al., 2000; Coppolino et al., 2001; Harrison et al., 2003; Bared et al., 2004). Proteomics Proteomics is the study of the structure, expression, local- ization, activity, interactions and cellular roles of all the proteins in a system (de Hoog and Mann, 2004). The degree to which we can comprehensively measure any of these parameters varies, with methods for determining expression and localization (Foster et al., 2006) being perhaps the most advanced. In the past, two-dimensional gel electrophoresis (2DGE) was used extensively to visu- alize a proteome. However, identification of more than a handful of the hundreds of spots from a 2DGE experiment has not proven very efficient, leading to nanoflow high- performance liquid chromatography\/tandem mass spec- trometry (LC-MS\/MS) surpassing 2DGE as the method of choice for most applications. Commercially available mass spectrometers and informatic tools for interpreting mass spectra have advanced to the point where labora- tories experienced in LC-MS\/MS can routinely and reli- ably identifying hundreds or thousands of proteins in exceedingly complex mixtures (Andersen et al., 2002; Foster et al., 2003; 2006). Although not yet as widely used, methods also exist for performing quantitative LC-MS\/MS experiments (Gygi et al., 1999; Ong et al., 2002; 2003). Proteomic characterization of biochemically enriched phagosomes Proteomic characterizations of phagosomes was initiated over a decade ago, as Desjardins et al. (1994a) devel- oped a method of isolating phagosomes containing inert, low-density particles by ultracentrifugation on a sucrose density gradient. During two initial studies (see Table 1 for a summary of all studies), 2DGE was utilized to resolve proteins from latex bead-containing phagosomes isolated from two human and mouse macrophage cell lines (U937 and J774 respectively), as well as one hamster and one rat kidney cell line (BHK and NRK respectively; Desjardins et al., 1994a,b). Proteins were then identified by comigra- tion with spots from a human keratinocyte-2D gel protein database and by Western blotting. Although these studies identified only a limited set of phagosomal proteins, con- servation between species was shown, and changes in spot intensities over 24 h of phagosome maturation revealed the dynamic nature of the phagosome proteome during this process. Using the same isolation approach, Garin et al. combined 2DGE with protein identification by mass spectrometry to characterize over 140 proteins between 0 and 24 h after phagocytosis (Garin et al., 2001). Proteins identified included cell surface proteins, subunits of the vacuolar ATPase, vesicular fusion and fission machinery, GTPases, hydrolases and other lyso- somal proteins, endosomal and exosomal markers, several cytoskeletal and coat proteins, ER proteins and mitochondrial proteins. Furthermore, the observed sequential delivery of hydrolases to phagosomes sug- gested heterogeneity in phagosomes and lysosomes and multiple interactions between these compartments. Subsequently, several studies have embarked on pro- teomic characterizations of phagosomes isolated from diverse species. In two independent reports, Soldati et al. characterized phagosomes from the amoeba Dictyostel- ium discoideum using a modified version of the isolation procedure described by Desjardins et al. (1994b). In these studies the preparation was treated with ATP to loosen the actin meshwork surrounding newly formed phagosomes and thus improve purity (Gotthardt et al., 2002; 2006). They identified 179 potential phagosome proteins with a similar array of functions to those previ- ously described. Importantly, based on changes in spot and band intensities on 2D gels and Western blots, they observed that over a maturation time of 165 min, proteins could be grouped into five major clusters: those that show a distinct peak at one time point, those that show constant abundance throughout, and those that show complex pat- terns and peak multiple times (Gotthardt et al., 2006). This suggested that linear maturation is an oversimplification and that there is either significant cross-talk between endocytic organelles or that alternative parallel pathways exist for maturing phagosomes. Nozaki and colleagues identified similar classes (early, intermediate, late and biphasic profiles) of proteins in Entamoeba histolytica trophozoites by LC-MS\/MS (Okada et al., 2005; 2006) using carboxylated latex-bead containing phago- somes isolated on a sucrose gradient. Using the semi- quantitative sequence coverage parameter they were able to track the relative abundances of proteins after 0, 30, 60 and 120 min of maturation. Recent improvements in the sensitivity and resolution of commercial mass spectrometers, as well as advances in quantitative proteomic techniques, have enabled the 1406 L. D. Rogers and L. J. Foster \u00a9 2008 The Authors Journal compilation \u00a9 2008 Blackwell Publishing Ltd, Cellular Microbiology, 10, 1405\u20131412 Ta bl e 1. Ch ro no lo gi ca ls u m m a ry o fp ha go so m al pr ot eo m ics st ud ie s to da te . R ef er en ce O rg an ism \/c el lt yp e Pr ot eo m ic m e th od Pr ot ei ns ID d\/ qu an tifi ed a M at ur at io n tim e (m in) b Qu an tifi ca tio nc Pa rti cl ed D es jar din se ta l. (19 94 a) M ou se \/m ac ro ph ag e 2D G E < 10 \/3 0. 5\/ 0, 1\/ 1, 1\/ 6, 1\/ 20 (h) Sp ot in te ns ity e 0. 8 mm LB f D es jar din se ta l. (19 94 b) H um an ,r a t, ha m st er , m o u se \/m ac ro ph ag e, ki dn ey 2D G E < 20 \/3 1\/ 0, 1\/ 1, 1\/ 6, 1\/ 12 ,1 \/2 4 (h) Sp ot in te ns ity 0. 8 mm LB G ar in e ta l. (20 01 ) M ou se \/m ac ro ph ag e 2D G E, M S > 14 0. 5\/ 0, 1\/ 1, 1\/ 24 (h) Sp ot in te ns ity 0. 8 mm LB Pi za rro -C er da e ta l. (20 02 ) H um an \/e pi th el ia l 2D G E, M S u n kn ow n 30 n \/a In lA -a n d In lB -c oa te d LB Ko v\u00e1 r\u030co v\u00e1 e ta l. (20 02 ) M ou se \/m ac ro ph ag e 2D G E, M S 6\/ no ne 2 h\/ 48 h n \/a F. tu la re ns is G ot th ar dt e ta l. (20 02 ) D ic ty os te liu m di sc oi de um \/a m o e ba 2D G E u n kn ow n 15 \/1 5 n \/a 0. 8 mm LB O ka da e ta l. (20 05 ) En ta m oe ba hi st ol yt ica \/tr op ho zo ite LC -M S\/ M S 85 n \/a n \/a ca rb ox yla td LB M ar io n e ta l. (20 05 ) En ta m oe ba hi st ol yt ica \/tr op ho zo ite 2D G E a n d LC -, M S\/ M S ~5 00 \/n on e 15 n \/a 2. 8 mm M B Bu rla k e ta l. (20 06 ) H um an \/n eu tro ph il Pr ef ra ct io na tio n, LC -M S\/ M S a n d 2D G E, M S 19 8 30 n \/a 2. 0 mm LB G ot th ar dt e ta l. (20 06 ) D ic ty os te liu m di sc oi de um \/a m o e ba 2D G E, M S 17 9\/ 92 5 5\/ 0, 15 \/0 , 15 \/1 5, 15 \/4 5, 15 \/1 05 , 15 \/1 65 Sp ot in te ns ity 0. 8 mm LB Ja co bs e ta l. (20 06 ) Te tra hy m en a th er m op hi la \/p ro to zo an Pr ef ra ct io na tio n, LC -M S\/ M S 73 n \/a n \/a 2. 0 mm re d- flu or es cin g PS O ka da e ta l. (20 06 ) En ta m oe ba hi st ol yt ica \/tr op ho zo ite LC -M S\/ M S 15 9\/ 15 9 0\/ 0, 5\/ 30 , 5\/ 60 ,5 \/1 20 Se qu en ce co ve ra ge g ca rb ox yla td LB St ua rt e ta l. (20 07 ) D ro so ph ila m e la no ga st er \/ e m br yo ni c- ha em at oc yt e Pr ef ra ct io na tio n, LC -M S\/ M S 61 7 n \/a n \/a 0. 8 mm LB R og er s a n d Fo st er (20 07 ) m o u se \/m ac ro ph ag e LC -M S\/ M S 38 2\/ 38 2 0\/ 0, 10 \/0 , 10 \/2 0, 10 \/3 0, 10 \/4 5, 10 \/6 0, 10 \/9 0, 10 \/1 20 SI LA C\u2013 st ab le is ot op es h 0. 8 mm LB Ju tra s e ta l. (20 07 ) m o u se \/m ac ro ph ag e pr ef ra ct io na tio n, LC -M S\/ M S a n d 2D E, M S 16 7\/ 16 7 1\/ 1 (h) sp ot a n d pe ak in te ns ity i 0. 8 mm LB Bo et tn er e ta l. (20 08 ) En ta m oe ba hi st ol yt ica \/tr op ho zo ite LC -M S\/ M S u n kn ow n \/n on e 0\/ 0, 5\/ 0, 10 \/0 ,1 0\/ 60 n \/a 2. 7 mm ca rb ox yla te d M B a. Th e n u m be ro fp ro te in s id en tifi ed in th e st ud y\/ th e n u m be ro fp ro te in s qu an tifi ed in th e st ud y. N o de no m in at or va lu e in di ca te s th at n o qu an tifi ca tio n w a s do ne o r th e n u m be ro fp ro te in s qu an tifi ed w a s n o tr e po rte d. b. Ag e(s )o fp ha go so m es st ud ie d in m in ut es u n le ss o th er w is e st at ed .A fra ct io n (e. g. 15 \/4 5) in di ca te s a pu lse -c ha se e xp er im en t(e .g. w he re ce lls w e re e xp os ed to th e pa rti cle s fo r1 5 m in a n d th en th e pa rti cle s w e re w a sh ed a w a y a n d m a tu ra tio n w a s a llo w ed to pr oc ee d fo r4 5 m in ). c. Qu an tita tiv e pr ot eo m ic m e th od u se d. Te ch ni ca la cc u ra cy o fm e th od s: st ab le is ot op es > pe ak \/s po ti nt en si ty > se qu en ce co ve ra ge . d. Pa rti cl e ph ag oc yt os ed . e. A co m pa ris on o ft he in te ns ity o fc o rr e sp on di ng sp ot s be tw ee n tw o 2D G E. f. LB :l at ex be ad .D is ta nc e in di ca te d th e a ve ra ge di am et er o fb ea ds u se d. g. A co m pa ris on be tw ee n tw o sa m pl es o ft he fra ct io n o ft he w ho le pr ot ei n se qu en ce o bs er ve d by ta nd em m a ss sp ec tro m et ry . h. St ab le Is ot op e La be llin g by a m in o a ci ds in ce ll cu ltu re .S ta bl e is ot op e m e th od s a re th e m o st a cc u ra te qu an tifi ca tio n st ra te gi es in pr ot eo m ics . i. Co m pa ris on o ft he pe ak vo lu m e in o n e LC -M S\/ M S a n a lys is w ith th an in a n o th er . 2D G E, 2- di m en sio na lg el e le ct ro ph or es is; M S, m a ss sp ec tro m et ry ;L C- M S\/ M S, liq ui d ch ro m at og ra ph y- co up le d ta nd em m a ss sp ec tro m et ry ;n \/a ,n o ta pp lic ab le . Proteomics and phagosome maturation 1407 \u00a9 2008 The Authors Journal compilation \u00a9 2008 Blackwell Publishing Ltd, Cellular Microbiology, 10, 1405\u20131412 simultaneous identification and quantification of several hundred proteins. Following isolation of latex bead- containing phagosomes from Drosophila S2 cells, Stuart et al. identified 617 potential phagosomal proteins and added 50 to 80 secondary components by looking at the phagosomal proteins in the context of an established protein\u2013protein interaction network (Stuart et al., 2007). In addition, they also used RNAi and fluorescence-based cell sorting to screen 837 genes for their role in phagocy- tosis of Staphylococcus aureus and Escherichia coli. This effort identified great variation in genes important for the uptake of each pathogen and was overlaid on the protein\u2013 protein interaction network to verify proteins that had a function in particle internalization. From this work, tubulin, Rab-GDI and chaperonin-containing T-complexes, as well as several components of the exocyst complex, emerged as being critically important for the internalization of bacteria. Using stable isotope labelled mouse macroph- ages, we have recently isolated phagosomes derived from the internalization of IgG-opsonized latex beads from a sucrose density gradient. The phagosomal proteome was investigated at high temporal resolution of (0, 10, 30, 30, 45, 60, 90 and 120 min) using stable isotope labelling by amino acids in cell culture (SILAC), which enables changes in the relative abundance of peptides to be deter- mined over time by LC-MS\/MS (Rogers and Foster, 2007). This resulted in the identification and quantification of 382 potential phagosome proteins based on dynamic profiles generated from the relative intensity of metaboli- cally labelled and unlabelled peptides. The proteins iden- tified were similar to those from previous studies and the quantitative profiles largely matched those previously characterized. However, consistent with data from work mentioned previously, several proteins displayed complex profiles peaking in abundance on phagosomes at several time points and supporting a more complex model of the maturation pathway, possibly involving heterogeneity between compartments and\/or multiple vesicle fusion events. Recently, Jutras et al. also utilized quantitative proteomics to analyse regulation of the phagosome pro- teome by interferon-g (Jutras et al., 2007), showing upregulation of most known phagosomal markers such as lysosomal hydrolases, vATPase subunits and several Rabs and SNAREs by this cytokine. Interstingly, while TLRs were also found to be upregulated on phagosomes following interferon-g treatment, receptors involved in phagocytosis of apoptotic cells (LRP\/CD91) and nutrient uptake (transferring receptor) were downregulated, sug- gesting modulation of phagocytic cargo during an immune response. Several studies addressing phagosomal proteomes in a range of organisms have now established a large degree of conservation within the pathway and identified a complex array of proteins as potentially or known to be associated with maturing phagosomes (Table 1). How- ever, while several of these have validated the presence of a select few proteins on phagosomes, differentiating between real phagosome players and contaminants is still an important issue. While it is not practical to verify each protein individually, alternative methods of isolating pha- gosomes should prove helpful in deciphering both false positives and false negatives. With the exception of mag- netic isolation of phagosomes from E. histolytica (Marion et al., 2005; Boettner et al., 2008), phagosomes from human neutrophils isolated on a continuous density gra- dient (Burlak et al., 2006), and the initial removal of the actin meshwork surrounding phagosomes in D. discoideum (Gotthardt et al., 2002; 2006), phagosome proteomics studies have all used the original enrichment procedure involving the isolation of low density latex beads on a sucrose density gradient. Sources of phagosomal membrane The surface area of a phagocytosing macrophage remains static, or if anything, it increases despite using an area of membrane equal to or greater than its surface area to engulf particles (Hackam et al., 1998; Holevinsky and Nelson, 1998), suggesting that intracellular mem- brane source(s) is required to replenish the PM and\/or to actually form the phagosome. In addition, during cross- presentation of antigens by phagocytic cells, the method of transporting antigenic peptides from the phagosomal lumen to the cytosol for subsequent transport into the ER, secretion and presentation on MHC Class I molecules is poorly defined (Huang et al., 1996; Rodriguez et al., 1999). Of all the proteomics-based discoveries addressing phagosome biology, the reports by Desjardins and col- leagues that the endoplasmic reticulum (ER) is directly involved in phagocytosis (Garin et al., 2001; Gagnon et al., 2002) have had the largest impact. Their work, and that of others (Guermonprez et al., 2003; Houde et al., 2003), presented a very attractive explanation for how antigens from intracellular pathogens can be transported from phagosomes for cross-presentation, and proposed the ER to directly contribute the vast majority of mem- branes used to construct the forming phagosome. More recently however, through extensive biochemical assays, fluorescent imaging, and electrom microscopy-based experiments, most of the lines of evidence in favour of ER-mediated phagocytosis have been challenged (Touret et al., 2005a,b). In addition to the plasmalemma, endoso- mal and post-Golgi membranes (Desjardins et al., 1994a; Jahraus et al., 1998), as well as exosomal membranes, have been shown to supply membrane to the forming phagosomal cup (Lee et al., 2007). 1408 L. D. Rogers and L. J. Foster \u00a9 2008 The Authors Journal compilation \u00a9 2008 Blackwell Publishing Ltd, Cellular Microbiology, 10, 1405\u20131412 Many groups, including those using proteomics, have consistently reported ER markers on phagosome prepa- rations (Garin et al., 2001; Gagnon et al., 2002; Touret et al., 2005a; Gotthardt et al., 2006; Rogers and Foster, 2007; Stuart et al., 2007), but until recently these reports were only qualitative. The ER-mediated model of phago- cytosis (Fig. 1) predicts that a very significant fraction of the phagosome membrane should be derived from the ER. No numbers are specified in the proposed model by Gagnon et al. but from the cartoon models based on electron microscopy images approximately 50% of the phagosomal membrane may come from the ER, with the remaining originating from the plasma membrane (PM; Gagnon et al., 2002). On the other hand, in the opposing model where the ER does not comprise the majority of forming phagosomes, the fraction of the phagosome com- prised of ER should be very small (Fig. 1). By fluorescent imaging Touret et al. have also shown 50% of GPI-YFP to be retained on the phagosomal membrane 5 min after particle internalization, suggesting that half the phagoso- mal membrane is comprised of plasmalemma at this time (Touret et al., 2005b). These observations and models then suggest a testable hypothesis: if the ER-mediated model is correct then the amount of PM on the phago- some, as a fraction of the total PM, should be about equal to the fraction of ER on the phagosome, after adjusting for the total surface areas of the PM and the ER. In a recent study, quantitative proteomics was used to use relative protein levels to estimate the percentage PM and ER membranes on phagosomes 10 min after internalization yielding approximately 10% and 0.2%, respectively, a ratio of 50:1 (Rogers and Foster, 2007). Thus, if there were 50 times more ER membrane than PM in a mac- rophage these data would support the ER-mediated phagocytosis model. However, the ER:PM ratio is prob- ably closer to 2:1 (Blouin et al., 1977), suggesting that the contribution of the ER in phagocytosis is negligible. There is little doubt, however, that some ER proteins are found on phagosomes (Gagnon et al., 2002; Touret et al., 2005a; Rogers and Foster, 2007). While not com- prising a sizable faction of the phagosomal membrane, it has been elegantly shown that these proteins function to modify and export phagosomal cargo into the cytosol for subsequent uptake in the ER and presentation by MHC Class I molecules (Guermonprez et al., 2003; Houde et al., 2003; Ackerman et al., 2006). While not yet addressing this issue directly, data from proteomic studies do support the cross-presentation competency of phago- somes, having identified MHC Class I molecules, com- ponents of the MHC class I loading complex, and protea- some subunits on phagosome preparations (Jutras and Desjardins, 2005; Burlak et al., 2006). Proteomics reports have also consistently identified mitochondrial proteins in phagosomal preparations (Garin et al., 2001; Burlak et al., 2006; Gotthardt et al., 2006; Okada et al., 2006; Rogers and Foster, 2007). Thus, small amounts of a select class of mitochondrial proteins may also comprise a portion of the phagosomal membrane, possibly to promote oxidative potential (Burlak et al., 2006; Okada et al., 2006). While these proteins may be delivered indirectly through other compartments (Neuspiel et al., 2008), Burlak and colleagues have confirmed the presence of some of these players by immunofluorescence microscopy (Burlak et al., 2006). Fig. 1. Models of phagosome formation and maturation. A. In the conventional model of phagocytosis (left side) the plasma membrane is the major source of membrane and the composition of the very early phagosome should be very similar to the plasma membrane. In the ER-mediated model (right side of figure) a large fraction of the phagosomal membrane is contributed directly from the ER. A significant prediction of this model is that the very early phagosome should contain a significant fraction of ER membrane. In both models other endomembranes, including the trans-Golgi network, are also likely to contribute membranes. B. In the conventional model of phagosome maturation (left side) the phagosome fuses sequentially with the early endosome, late endosome and lysosome. Two quantitative proteomic studies (Gotthardt et al., 2006; Rogers and Foster, 2007) have demonstrated that there are likely more distinct fusion events, presumably with subpopulations of the three main classes of endosomes. Proteomics and phagosome maturation 1409 \u00a9 2008 The Authors Journal compilation \u00a9 2008 Blackwell Publishing Ltd, Cellular Microbiology, 10, 1405\u20131412 The Holy Grail for phagosome proteomics: bacteria-containing vacuoles Opsonized latex beads are an excellent tool for exploring normal phagosome maturation; they are inert, easily manipulated and can be obtained in different sizes to mimic various targets a phagocyte is likely to meet. However, some of the most interesting aspects of phagosome matu- ration revolve around the abilities of intracellular patho- gens to bypass the normal maturation process. Two examples of many such mechanisms are seen in Salmo- nella enterica, which prevents formation of the phago- lysosome (Haraga et al., 2008), and Mycobacterium tuber- culosis, which blocks lumenal acidification by preventing conversion of Rab proteins between their GDP- and GTP- bound states (Deretic et al., 2006). Several groups have reported procedures for biochemical enrichment of bacteria-containing vacuoles (BCV) but no proteomic analysis of such a compartment has been reported (Kov\u00e1r\u030cov\u00e1 et al., 2002 analysed Francisella tularensis- containing phagosomes by 2DGE but only reported the identities of two host-derived proteins). We, and probably others, have expended significant effort to try to analyse the proteome of BCV, those containing S. enterica serovar Typhimurium in our case. The conventional sucrose density gradient approach has been unsuccessful because BCV are a very similar density to other compart- ments of the cell, most significantly mitochondria that probably share a common ancestry with bacteria (Margu- lis, 1968) and would thus have a similar density.Attempts to shift the density of BCVs by using latex nanoparticles or by making the BCVs magnetic by phagocytosing magnetic beads together with bacteria were not clean enough for analysis, and fluorescence-activated cell sorting of BCVs containing green fluorescent protein-expressing S. typh- imurium was not quick enough to obtain sufficient material of the unstable BCV. Efforts to alter the density of mito- chondria or immunodeplete them in the hopes of cleaning up that region of the gradient were successful in that the mitochondrial contamination was decreased but unfortu- nately this only revealed the presence of many other host-derived membranous compartments in higher abun- dance that the BCVs. Nonetheless, as current work unveils a vast array of different BCVs comprising few to several bacteria and varying greatly in size and likely also in content (Birmingham et al., 2008), the notion of puri- fying these vacuoles to analyse their composition and various functionalities becomes increasingly intriguing (Birmingham et al., 2008). Conclusions Global, unbiased proteomics approaches have made sig- nificant contributions to phagosome biology, perhaps more so than any other area of cell biology. Proteomics opened up the debate over the role of the ER in phago- cytosis (Garin et al., 2001) and more advanced quantita- tive methods can now be used to test hypotheses arising from this model (Rogers and Foster, 2007). A very com- prehensive systems biology approach to studying phago- somes demonstrated a role for the exocyst complex in phagosome maturation (Stuart et al., 2007). The use of more advanced quantitative techniques to evaluate the maturing phagosomal proteome revealed a role for het- erotrimeric G-proteins in phagosme maturation (Gotthardt et al., 2006) and has suggested that the classical model of three consecutive fusions events with different endosomal systems is probably overly simplistic (Gotthardt et al., 2006; Rogers and Foster, 2007). Phagosome proteomics studies typically use opsonized latex beads to model phagocytosis (for exception see Kov\u00e1r\u030cov\u00e1 et al., 2002) and for the normal process this is probably reasonable. However, one of the most inter- esting questions about phagocytosis is how certain in- tracellular pathogens are able to manipulate normal pha- gosome maturation in order to survive inside the host (e.g. S. enterica, M. tuberculosis). 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(2005a) The nature of the phagosomal membrane: endoplasmic reticulum versus plasmalemma. J Leukoc Biol 77: 878\u2013885. Touret, N., Paroutis, P., Terebiznik, M., Harrison, R.E., Trombetta, S., Pypaert, M., et al. (2005b) Quantitative and dynamic assessment of the contribution of the ER to phagosome formation. Cell 123: 157\u2013170. Underhill, D.M. (2005) Phagosome maturation: steady as she goes. Immunity 23: 343\u2013344. Vieira, O.V., Botelho, R.J., and Grinstein, S. (2002) Phago- some maturation: aging gracefully. Biochem J 366: 689\u2013 704. 1412 L. D. Rogers and L. J. Foster \u00a9 2008 The Authors Journal compilation \u00a9 2008 Blackwell Publishing Ltd, Cellular Microbiology, 10, 1405\u20131412 The dynamic phagosomal proteome and the contribution of the endoplasmic reticulum Lindsay D. Rogers and Leonard J. Foster* Centre for Proteomics, Department of Biochemistry and Molecular Biology, University of British Columbia, 301-2185 East Mall, Vancouver, BC, Canada V6T 1Z4 Edited by Emil R. Unanue, Washington University School of Medicine, St. Louis, MO, and approved October 4, 2007 (received for review June 23, 2007) Macrophages use phagocytosis to control the spread of pathogens in the body, to clear apoptotic cells, and to aid in tissue remodeling. The phagosomal membrane is traditionally thought to originate from the plasmalemma and then go through a series of maturation steps involving sequential fusion with endosomal compartments, leading to the formation of a phagolysosome. A recent model suggests that the endoplasmic reticulum (ER) is involved in the maturation as well. Here we use stable isotope labeling and multiple quantitative proteomic approaches to follow the dynamic composition of the maturing phagosome in RAW 264.7 macro- phage cells to a greater depth and higher temporal resolution than was previously possible. Analysis of the results suggests that the traditional model of a linear sequence of fusion events with different compartments is more complex or variable than previ- ously thought. By concomitantly measuring the degree to which each component is enriched on phagosomes, our data argue that the amount of ER involved in phagocytosis is much less than predicted by the model of ER-mediated phagocytosis. innate immunity \u0001 organelle \u0001 phagocytosis \u0001 stable isotope labeling \u0001 latex bead vacuoles Phagocytosis is the process by which cells engulf particles.Primitive eukaryotes use phagocytosis primarily to obtain nutrients (1), whereas in more complex organisms it serves additional functions such as the clearance of apoptotic cells and various pathogens (2). Internalized objects are contained in a membrane-bound vacuole called the phagosome, and current models have the phagosome traversing a complex maturation process involving sequential fusion with early endosomes (EE), late endosomes (LE), and ultimately with lysosomes (LS) (3, 4). In this model, full maturation is characterized by luminal acid- ification and acquisition of hydrolytic enzymes, which serve to degrade or kill the cargo, typically microbes, within 2\u20134 h after internalization (5). Previous studies have aimed to characterize the phagosome proteomes in cell lines from mouse and fruit f ly, as well as from Dictyostelium discoideum, Entamoeba histolytica, and Tetrahymena thermophila (6\u201310). Of particular note, the studies of Desjardins and colleagues (6) in mouse macrophages led them and others to propose a role for the endoplasmic reticulum (ER) in phagocytosis (11, 12), although this model has been challenged recently (13). Here we apply stable isotope labeling by amino acids in cell culture (SILAC) to develop a comprehensive, quantitative model of phagosome maturation that allows us to test several hypotheses suggested by current models of the process. Results and Discussion Maturation into a phagolysosome is absolutely crucial for even- tual destruction of phagocytosed objects, whereas the success of pathogens such as Salmonella enterica andMycobacterium tuber- culosis depends largely on their ability to avoid phagolysosomal killing. To gain a better understanding into the process of phagosome maturation we used latex beads to model phagocy- tosis in RAW 264.7 mouse macrophage cells and SILAC with an LTQ-Orbitrap to measure the dynamics of the maturing phago- somal proteome with unparalleled accuracy. IgG was chosen as an opsonin rather than whole serum (6) to reduce potentially confounding effects of phagocytosis through different receptor systems. IgG-opsonized latex beads were allowed to internalize for 10 min, and then latex bead-containing vacuoles (LBVs) were harvested at seven different time points (Fig. 1).We focused on events within 2 h of phagocytosis because most internalized objects are dead or destroyed by this time and there is no apparent physiological relevance to allowing vacuoles containing inert latex to mature for longer periods. From at least three biological replicates of each time point we identified 505 proteins associated with LBVs, 382 of which could be reliably quantified across various time points [supporting information (SI) Table 1]. Based on current models of phagosomematuration (4), we expected to find a large influx of EE markers, followed by LE markers and ending with a bolus of LS proteins. Certain markers of these compartments did peak at the expected time points, but endosomal proteins and the vesicle trafficking ma- chinery as a whole did not arrive in three discrete packages, as predicted. Biochemical enrichment of an organelle is never perfect, because complete separation from all other organelles is essen- tially never achieved. Given what is known about phagosome development, one can easily imagine a situation where the actin mesh surrounding LBVs at certain stages could entrap pieces of other organelles, leading to their apparent time-dependent copurification with LBVs. In our experimental approach, one would expect an unchanging SILAC profile over time if proteins were enriched at the same time as LBVs but independent of the LBV maturation itself. However, such is not the case for most markers of other organelles that we have measured here (SI Table 2). One possible explanation for this could be that some organelles get trapped to varying degrees with the LBVs (e.g., caught in the surrounding actin mesh) at certain time points. An alternative explanation could be that the cell lysis procedure disrupts LBVs themselves, leading to some nonspecific associ- ation of cytoplasmic proteins with the beads. Proteins from other organelles should generally not affect profiles of LBV proteins, however, except where a protein is shared between LBVs and the copurifying organelle. Multiple Visits to the LBV. One by one the movement of most known endosomal markers has been followed on maturing phagosomes using Western blotting (14). Our data set contains Author contributions: L.J.F. designed research; L.J.F. performed research; L.J.F. contributed new reagents\/analytic tools; L.D.R. and L.J.F. analyzed data; and L.D.R. and L.J.F. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Freely available online through the PNAS open access option. *To whom correspondence should be addressed. E-mail: ljfoster@interchange.ubc.ca. This article contains supporting information online at www.pnas.org\/cgi\/content\/full\/ 0705801104\/DC1. \u00a9 2007 by The National Academy of Sciences of the USA 18520\u201318525 \u0001 PNAS \u0001 November 20, 2007 \u0001 vol. 104 \u0001 no. 47 www.pnas.org\u0002cgi\u0002doi\u000210.1073\u0002pnas.0705801104 the majority of proteins these early studies focused on, and the profiles measured by SILAC largely agree with those reported previously. For instance, EE antigen 1 is most abundant at 30 min, Niemann\u2013Pick C1 and charged multivesicular body protein 6 (CMVB6) peak around 60 min, and the LS-associated mem- brane proteins (LAMPs) and cathepsins peak at 90 or 120 min (Fig. 2). However, we also observed that many proteins, includ- ing LAMP-1, heterotrimeric G proteins from the plasma mem- brane, and some cathepsins, display a biphasic profile with a smaller peak earlier in the time course. Although not explicitly Plasma Membrane 0 10 Internalization Maturation 20 30 45 60 90 120 Expt 1 Arg6\/Lys4 Arg10\/Lys8 Arg0\/Lys0 Expt 2 Arg0\/Lys0 Arg4\/Lys6 Arg10\/Lys8 Arg0\/Lys0 Arg6\/Lys4 Arg10\/Lys8Expt 3 Y Y Y Y Y Y Y Y 0 65000 130000 519 520521 522 523 524 0 65000 130000 519 520 521 522 523 524 0 65000 130000 519 520 521 522 523 524 In te n si ty 0 65000 130000 a b c Fig. 1. Applicationof SILAC tophagosomematuration. (a) IgG-opsonized, 0.8-\u0001mlatexbeadswereadded toRAW264.7 cells (T\u00010) andallowed tophagocytose for 10 min, at which point external beads were washed away and LBVs were allowed to mature for the times indicated by dashed gray lines. Because SILAC is limited to a maximum of three conditions per experiment, one time point in each experiment was used to scale the other time points across experiments as described inMaterials and Methods. For instance, the 90-min time point was used to scale between experiments 2 and 3 in this example. (b) Measured spectra for the [M\u0002 2H]2\u0002 ion of ATIGADFLTK from Rab7 covering all seven time points in three experiments are shown. The abscissa scale for each peak cluster ism\/z, butnote that the three clusters ineach rowarenotorderedbym\/zbut ratherby the timepoint that they represent. Theordinateaxesoneachhavebeencorrected by the isotope enrichment factor measured for each experiment. (c) The overall profile that would be calculated for ATIGADFLTK based on only the three experiments shown. In reality the profile for a protein was the averaged, normalized intensity at each time point for each peptide identified from that protein. Time (min) L o g 10 (R el at iv e ab u n d an ce ) 0.3 -0.3 -0.6 10 20 30 45 60 90 120 CathB CathS CathZ LAMP2 CathA LAMP1 NPC1 EEA1 CMVB6 G\u03b21 Go\u03b12 0 10 20 30 45 60 90 120 - CathA Fig. 2. Endosomal proteins repeatedly move on and off LBVs. LBV profiles of several endosomal and plasma membrane proteins identified in this study are shown. Cath, cathepsins; NPC1, Niemann\u2013Pick C1 protein; CMVB6, charged multivesicular body protein 6. Shown are average profiles from at least three replicates of each time point. (Inset) Western blot of CathA across the time points indicated. Rogers and Foster PNAS \u0001 November 20, 2007 \u0001 vol. 104 \u0001 no. 47 \u0001 18521 CE LL BI O LO G Y defining a biphasic profile, different studies have found LAMP-1 on phagosomes very early after phagocytosis (15) or at later times (4). Many of the other proteins we measured also display a similar pattern (Fig. 2 and SI Table 2), suggesting that the EE, LE, and LS designations are too simplistic and that the concep- tualized EE, LE, and LS compartments contain significant heterogeneity. Although there is undoubtedly some \u2018\u2018spillage\u2019\u2019 or leakiness of even classical marker proteins such as LAMP-1 into different compartments, this alone is insufficient to explain the patterns we observe. The biphasic profiles observed are unlikely to be artifacts of nonsynchronous internalization, the LBV isolation procedure, or the quantitative MS approach for several reasons: (i) timing of washes and harvesting varied by no more than 3%, (ii) some of the profiles are confirmed byWestern blots (Figs. 2 and 3), (iii) the peaks and troughs of all of the profiles do not align, and (iv) not all proteins show the biphasic profile. One of the hallmarks of phagosome maturation is the steady decline in the luminal pH, brought about by the acquisition of the vacuolar ATPase (vATPase). Many subunits of the vATPase were measured in this study, and as a group they peak at 90 min (SI Fig. 6), as predicted. However, the vATPase subunits all show a biphasic profile as well, with an earlier peak or plateau at 30 or 45 min. This suggests that the maturing phagosome initially fuses with an LE- or LS-like compartment, which is then followed by a larger influx of LE\/LS proteins. These conclusions are also supported by the LBV profiles of the proteins with a mechanistic role in vesicle traffic, the SNARE and Rab families of proteins. Participation of SNAREs such as syntaxin 7 and vesicle-associated membrane protein 8 (VAMP8)\/endobrevin in LE\/LS fusion is well known (16), and their LBV profiles reflect this action. Intriguingly, though, several SNAREs (Fig. 3 and SI Table 2), including VAMP8, also show a biphasic profile. There is still much debate as to whether SNAREs alone are sufficient to dictate fusion of two specific membranes (17); regardless, the biphasic profile of several SNARE proteins suggests that certain classes of compartments are fusing with the maturing LBV more than once. Rabs. A number of Rab guanine triphosphatases are known to localize to specific compartments in the endocytic pathway and regulate membrane traffic (18). Our proteomic approach iden- tified 20 Rabs on LBVs, 16 of which could be quantified sufficiently to construct a time course. Of these 16, 12 were also among the 48 Rabs examined for their phagosomal association by Smith et al. (19). As shown in Fig. 4, the mass spectrometry and microscopy profiles were largely congruous, at least with respect to whether a protein was increasing or decreasing in abundance at a certain time, if not in the actual magnitude. There were a couple of notable exceptions, particularly Rabs 14, 27, and 35, where our approach detected a decreased level of association in the middle time points relative to that measured by Smith et al. (19), possibly because of differences in the Salmonella-containing vacuole versus IgG-opsonized LBVs or epithelial cells versus macrophages. Nonetheless, the LBV pro- files of these and the other Rabs measured here support the general conclusion that maturation involves more than just three discrete fusion events. At least one Rab protein peaked in L o g 10 (R el at iv e ab u n d an ce ) Time (min) 10 20 30 45 60 90 120 -0.4 0 0.4 SNAP23 Sxn7 VAMP4 Vti1b NSF SNAP29 VAMP8 10 20 30 45 60 90 120 - VAMP4 10 20 30 45 60 90 120 NSF - Fig. 3. Dynamic profiles of SNAREs. Shown are LBV profiles of several SNARE and SNARE-associated proteins identified in this study. Sxn7, syntaxin 7; SNAP23 and SNAP29, synaptosome-associated protein of 23 or 29 kDa. Shown are average profiles fromat least three replicates of each time point. (Insets)Western blots of VAMP4 and NSF across the time points indicated. Rab34 150 -150 Rab32 150 -150 Rab27A 150 -150 Rab18 150 -150 Rab14 150 -150 Rab11B 150 -150 Rab10 150 -150 Rab7 150 -150 Rab5C 150 -150 Rab5B 150 -150 Rab1A 150 -150 150 -150 180120600 Rab35 Time (min) Fig. 4. Dynamic profiles of Rabs. Shown are profiles of the 12 Rab proteins common to the LBVs in our study and the Salmonella-containing vacuoles of Smith et al. (19). The data for our data (solid lines) and Smith et al.\u2019s data (dashed lines) plots are normalized to the first time point (10min for our data and15min for thedataof Smithet al.), log10-transformed, and thenexpressed as a percentage of the highest absolute measurement. Our data profiles are from at least three replicates of each time point. 18522 \u0001 www.pnas.org\u0002cgi\u0002doi\u000210.1073\u0002pnas.0705801104 Rogers and Foster abundance at each time measured, except for 45 min, suggesting that there are at least five discrete events or, more likely, a continuum of fusion. Unknown Players in Phagosome Maturation. Although we under- took this study to try to address some hypotheses about phago- some maturation, the depth and temporal dimension of our data set also generate testable hypotheses by describing the phago- somal dynamics of several proteins not previously predicted to play a role in this process. To pick two of many examples, annexins A2 and A7 were enriched on LBVs at 60 min (SI Table 2), suggesting that they arrive with LE or play a role in their fusion with LBVs. Annexin A2 was previously shown to facilitate endosome fusion (20) and biogenesis (21), whereas A7 may be regulated byMycobacterium avium (22). BecauseMycobacterium is known to prevent phagolysosomal fusion, our data suggest that annexin A7 could be tested as a potential host target of Mycobacterium effectors. Likewise, annexin A2 could be tested as a more general regulator of LE traffic. The Contribution of the ER. ER proteins in biochemically enriched phagosomes were typically written off as contaminants until Desjardins and colleagues (11) used electron microscopy to argue that the ER itself could phagocytose particles and that successive waves of ER interact with the maturing phagosome (11). The role of the ER in phagocytosis is far from resolved, however, because more recently Grinstein and colleagues (23) proposed a diametrically opposed model where the ER plays at most a very small role in phagosome maturation, leaving no apparent middle ground to incorporate both theories. Several testable hypotheses come out of this controversy; here we use proteomic analysis of LBV preparations to address some of them. The ER is typically quite dense (24), so it should not migrate at all close to latex beads in a density gradient. However, the ER forms a reticular network throughout the cytoplasm, so if ER membrane is not contiguous with the phagosomal membrane then it is plausible that some of the ER gets trapped in the actin mesh surrounding the LBV or is otherwise physically but not functionally associated with vacuoles. To address the possibility that ER proteins are not integral to the phagosomal membrane, we quantified the effect of a pH 11.5 buffer on components of the LBV purification. High pH is commonly used to strip away nonintegral membrane proteins (25), so if pieces of the ER were somehow contaminating the LBV preparation and not part of the LBVs themselves then ER proteins would be expected to be depleted in LBVs isolated in high pH. However, most common ERmarker proteins, with the exception of calnexin, were present at equal levels in LBVs isolated at physiological pH and at pH 11.5, as were integral LBV membrane proteins such as the Fc\u0002 receptor and the cation-dependent mannose 6-phosphate recep- tor (Fig. 5a). This observation supports the conclusions of Garin et al. (6) that ER proteins are integral to the vacuolar membrane. That calnexin is increased with high pH suggests that a fraction of it may reside on some ER membrane not integral to the phagosomal membrane. The surface area of an actively phagocytosing macrophage stays approximately constant, or, if anything, it increases, despite PDIA3 PDIA4 Calnexin Sec61 CD-M6PR LAMP2 EEA1 Calreticulin Fc\u03b3R Rab5C g o L 01 ) V B L n o lat ot f o n oitcar F( Protein # 1 300 CathA CathZ Rab5B Rab7 TLR7 Na\/K\u03b23 PDIA3 ERp29 Calnexin Proteins of the large and small ribosomal subunits CD-M6PR EEA1 Calreticulin Fc\u03b3R g o L 01 )s V B L la mr o n\/11 H p( Protein # 1 200 NSF \u03b1SNAP vATPase -2 0 2a b Gi\u03b12 G\u03b22 -4 -2 Fig. 5. ER proteins on LBVs are integral to the membrane but constitute only a small fraction of the total ER. (a) Ranked relative abundances of proteins on LBVs isolated from RAW cells at pH 11.5 versus at pH 7.2. Specific ribosomal proteins (blue), ER markers (red), integral LBV membrane proteins (red), and membrane-associatedproteins (green) are indicated. PDIA3, protein disulfide isomerase 3;\u0003SNAP, solubleNSF attachment protein; Fc\u0002R, Fc\u0002 receptor; CD-M6PR, cation-dependent mannose 6-phosphate receptor. (b) The amount of each protein found on LBVs at 10 min, expressed as a fraction of the total amount of that protein in the cell normalized for the fraction of cells taking up beads. Specific ERmarkers (red), endosomal\/phagosomalmarkers (black), and plasmamembrane markers (green) are indicated. TLR7, Toll-like receptor7; Cath, cathepsin;Na\/K\u00043,Na\u0002\/K\u0002-ATPase\u00043 subunit. The ratios are cutoff at 100-foldupordownbecause the linearity of the signal in the Orbitrap starts to drop off beyond this. Rogers and Foster PNAS \u0001 November 20, 2007 \u0001 vol. 104 \u0001 no. 47 \u0001 18523 CE LL BI O LO G Y needing to use an area of membrane equal to or greater than its surface to engulf the particles (26, 27). This suggests that an intracellular source of membrane is used in replenishing the plasma membrane (PM) or in actually forming the phagosome. Endosomal and post-Golgi membranes are known to be such sources (14, 28), and Desjardins and colleagues (11) have proposed that the ER may be directly involved in phagocytosis. The area of the ERmembrane is approximately twice that of the PM in hepatic phagocytes (Kupfer cells) (29), so it is reasonable to assume that the ratio would be similar in RAW cells. For each of\u0003300 proteins found on LBVs, wemeasured the portion of the total complement of that protein actually found associated with the vacuole. Approximately 10% of the PM was found on LBVs, based on the enrichment of several PM markers (Fig. 5b), and, given the assumed 2:1 ratio of ER:PM membrane, the ER- mediated phagocytosis model would predict at least 20% of the ER membrane to be present on LBVs. However, LBV prepa- rations contained only \u00040.3% of the cellular total of five ER marker proteins used by others (11, 23): protein disulfide isomerases, calnexin, calreticulin, and Sec61 (Fig. 5b). These data argue against the ER being a major source of membrane for newly forming phagosomes, whether it is through direct inter- nalization into the ER (11) or as a source for replenishing lost PM. Our data do not, however, address the observation that the ER may be involved in engulfing only large particles (30) or the possibility that replenishment from the ER is required only after long, sustained periods of phagocytosis. Conclusions The dynamic profiles of 382 proteins associated with biochemically enriched phagosomes from RAW cells described here provide a comprehensive view of the process of phagosome maturation at a temporal resolution exceeding that of most previous studies focus- ing on even single phagosomal proteins. Our data are largely congruous with the few knownmarkers of the process, but they also suggest that maturation does not just proceed as three discrete fusion events with EE, LE, and then LS. By directly measuring enrichment of proteins on LBVs, our data shed light on the current controversy surrounding the role of the ER in phagocytosis (11, 13) by arguing strongly against the ER-mediated phagocytosis model. Our data enhance the knowledge of the latex bead model of phagocytosis and will hopefully open new avenues for understand- ing pathogen-containing vacuoles. Materials and Methods Cell Culture and SILAC. RAW 264.7 mouse macrophage-like cells weremaintained as described (23) and split at a 1:4 dilution into one of three SILACmedia formulations: (i) normal isotopic abundance arginine (42 mg\/liter) and lysine (73 mg\/liter), (ii) [13C6]arginine (43.5 mg\/liter; Cambridge Isotope Laboratories, Andover, MA) and [2H4]lysine (75 mg\/liter), or (iii) [13C6,15N4]arginine (44.5 mg\/liter) and [13C6,15N2]lysine (77mg\/liter). All SILACmedia were based on arginine- and lysine-free Dulbecco\u2019s modified Eagle\u2019s medium (CaissonLabs,North Logan,UT) supplementedwith 10% heat-inactivated, dialyzed FBS (Invitrogen), 1% L-glutamine, and 1% penicillin\/streptomycin (ThermoFisher Scientific, Bremen, Germany). Cells were passaged three more times in the above media at a 1:4 dilution each time before use. In our experience these labeling conditions led to 100% incorporation inmost cell types, but in RAW cells we found inconsistent levels of heavy isotope incor- poration from experiment to experiment. We attribute this phe- nomenon to the phagocytic capacity of these cells, which likely allows them to scavenge substantial levels of amino acids from the unlabeled proteins in serum. To compensate for incomplete label- ing we analyzed 2 \u0001g of the combined lysates from all three label sets and used the incorporation levels measured in the lysate to correct the ratios measured in each experiment individually. This incorporation patternmeant that the ion intensity for the light form of each SILAC triplet was typically higher than for the two heavier forms. Accordingly, to avoid a bias in the data-dependent acqui- sition the time point used for the light form was rotated from experiment to experiment. LBV Isolation. Six 14-cm plates of RAW cells were used per time point in each experiment. To initiate internalization the growth media were removed, the cells were washed once in PBS, and then 10 ml of serum-free and arginine\/lysine-free DMEM was added to each plate. Mouse Ig-opsonized 0.8-\u0001m latex beads (Sigma-Aldrich) were added to the cells at a 1:400 dilution and allowed to incubate for 10 min, at which time the bead solution was aspirated, the cells were washed once with PBS, and then 12 ml of the appropriate SILAC media was added back for various lengths of time. Each time point was measured between three and six times. For normal time courses LBVs were isolated essentially as described, using only the 10%, 25%, and 35% layers on the discontinuous sucrose density gradient (31). The same protocol was used to test the effects of high pH on LBVs except that 100 mMNa2CO3 (pH 11.5) was used to lyse the cells instead. LC\/MS. Protein samples were solubilized in 1% sodium deoxy- cholate and 50 mM NH4Cl, heated to 99\u00b0C for 5 min, and then reduced, alkylated, digested, and analyzed on a linear trapping quadrupole-Orbitrap mass spectrometer (ThermoFisher Scien- tific) as described (32). Formaldehyde Labeling. Formaldehyde isotopologues (33) were used in place of SILAC to quantify the effects of high pH on LBVs and the degree of enrichment of LBV components. Briefly, peptides purified with C18 Stage Tips (34) were resus- pended in 5 \u0001l of 200 mM formaldehyde or deuterated form- aldehyde (Cambridge Isotope Laboratories) and 0.5 \u0001l of 1 M sodium cyanoborohydride and incubated for 30 min at room temperature away from light. The reaction mixture was then adjusted to pH 7.5, and a further 5 \u0001l of the respective formaldehyde isotopologue plus 1 \u0001l of 1 M cyanoborohydride was added. The reaction was allowed to continue for a further 30 min before being quenched by addition of 6 \u0001l of 2.5 M NH4Cl for 10 min at room temperature. Data Analysis. Fragment spectra were extracted as described (32) and searched against the mouse International Protein Index data- base supplemented with the sequences of all human keratins and abundant bovine serum proteins (v3.25; 52,434 sequences) using Mascot (v2.1; Matrix Science) and allowing only tryptic peptides with up to one missed cleavage. MSQuant (http:\/\/msquant.source- forge.net) was used to parse Mascot result files, to recalibrate mass measurements, and to extract quantitative ratios (SI Table 2). The final list of proteins was generated by using finaList.pl, an in-house script available upon request. A false discovery rate for protein identifications based on two or more peptides (SI Table 1) with a measured mass accuracy \u00055 ppm (the overall average was 0.56 ppm), a Mascot score\u000526, and length of at least eight residues was estimated to be\u00050.5% using reversed database searching. For Fig. 4, previously reported phagosomal association curves for Rab proteins with \u0006invA\/Inv-containing vacuoles were extracted from the original publication (19) using Photoshop (Adobe Systems) to measure the density of the gray boxes for each time point. Rabbit antibodies against CathA and VAMP4 and mouse antibodies against N-ethylmaleimide-sensitive factor (NSF) were purchased from Abcam. We thank Brett Finlay, Nat Brown, Erin Boyle, Carmen de Hoog, and Brian Coombes for helpful discussions and advice. This work was supported by Canadian Institutes of Health Research Operating Grant MOP-77688 (to L.J.F.). 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Rogers and Foster PNAS \u0001 November 20, 2007 \u0001 vol. 104 \u0001 no. 47 \u0001 18525 CE LL BI O LO G Y","attrs":{"lang":"en","ns":"http:\/\/www.w3.org\/2009\/08\/skos-reference\/skos.html#note","classmap":"oc:AnnotationContainer"},"iri":"http:\/\/www.w3.org\/2009\/08\/skos-reference\/skos.html#note","explain":"Simple Knowledge Organisation System; Notes are used to provide information relating to SKOS concepts. 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