Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Quantitative mass spectrometry to discover interactors of parkin E3 ubiquitin ligase, a protein implicated… Pu, Christopher Hao 2009

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
24-ubc_2010_spring_pu_hao.pdf [ 11.25MB ]
Metadata
JSON: 24-1.0068961.json
JSON-LD: 24-1.0068961-ld.json
RDF/XML (Pretty): 24-1.0068961-rdf.xml
RDF/JSON: 24-1.0068961-rdf.json
Turtle: 24-1.0068961-turtle.txt
N-Triples: 24-1.0068961-rdf-ntriples.txt
Original Record: 24-1.0068961-source.json
Full Text
24-1.0068961-fulltext.txt
Citation
24-1.0068961.ris

Full Text

Quantitative Mass Spectrometry to Discover Interactors of Parkin E3 Ubiquitin Ligase, a Protein Implicated in Early-Onset Parkinson’s Disease  by  Christopher Hao Pu B.Sc.H., University of British Columbia, 2007  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Biochemistry and Molecular Biology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  December 2009  © Christopher Hao Pu, 2009  ABSTRACT Ubiquitylation is a major post-translational modification based on a network of about six hundred E3 ubiquitin ligases in human. It is involved in several processes such as proteolysis, vesicle trafficking and DNA damage response. Mutations in PARK2, which encode parkin E3 ubiquitin ligase, account for half of autosomal recessive juvenile Parkinsonism cases, an early onset form of Parkinson’s disease. Multiple PARK2 mutations underlie the RING domain, which contains ligase activity. This finding suggests an inability for substrate ubiquitylation may trigger neurodegeneration. We used a quantitative proteomics approach to seek identifying parkin substrates and interactors. We first developed and tested new methods to enrich for ubiquitylated proteins that could potentially be used to study the influence of parkin on the ubiquitin proteome. In our first approach, ubiquitin conjugates were purified from SH-SY5Y neuroblastoma expressing His8-biotin-ubiquitin by tandem affinity purification. A second approach to purify ubiquitylated proteins was based on affinity chromatography using S5a proteasome receptor that bound to poly-ubiquitylated proteins. We determined that both approaches were not adequate for identifying low abundance parkin substrates. We then sought to identify which proteins were associated with parkin. Parkin interactors were enriched from SH-SY5Y expressing FLAG-parkin versus endogenous parkin by anti-FLAG immunoprecipitation in the context of SILAC. Proteins from the neuroendocrine chromogranin-secretogranin family were highly enriched suggesting a potential granin vesicle trafficking role for parkin. CCCP, a mitochondrial uncoupling agent was also employed to investigate parkin ligase interactors during mitochondrial stress since parkin localizes to mitochondria to promote mitophagy upon a reduction in ii  mitochondrial membrane potential. Several actin related proteins were enriched from FLAG-parkin cells treated with CCCP including non-muscle unconventional signaling myosin suggesting a potential role for these proteins during parkin-mediated mitophagy.  iii  TABLE OF CONTENTS Abstract……………………………………………………………………………………...……ii Table of Contents……………………………………………………………………………….iv List of Tables……………………………………………………………………………………vii List of Figures………………………………………………………………………….………viii List of Abbreviations…….………………………………………………………………….......ix Acknowledgements…………………………………………………………………………….xii CHAPTER 1. INTRODUCTION………………………………………………………………..1 1.1 The ubiquitin system……………………………………………………………….2 1.2 The UPS in Parkinson’s disease..………………………………………………..3 1.3 Parkin and the mitochondrial connection………………….…………………….8 1.4 Methods for the purification of ubiquitylated proteins…………………………11 1.4.1 Purification of ubiquitylated proteins from yeast…………..………..12 1.4.2 Purification of ubiquitylated proteins from mammalian cells……….12 1.5 Project Aim: Identification of novel parkin E3 ubiquitin ligase substrates and interacting proteins at the basal level and upon mitochondrial stress…………...13 CHAPTER 2. MATERIALS AND METHODS……………………………………………….14 2.1 Mammalian expression construct cloning……………………………………...15 2.1.1 His8-biotin-ubiquitin..…………………………………...……...……….15 2.1.2 His8-ubiquitin………...………………………...………………………...16 2.1.3 Myc-parkin………………………………….....………………………..16  iv  2.2 Cell culture techniques.……………………..…………………… .…………………….16 2.2.1 Cell maintenance…...……………………………...…………………..17 2.2.2 DNA transfection…...……………………………...…………..……….17 2.2.3 Generation of stable cell lines……………...…………………....……18 2.2.4 SILAC…………..……………………...………………………….…….18 2.3 Preparation of biochemical purification techniques.…………………………..19 2.3.1 GST-S5a sepharose…………………………...…………...………….19 2.3.2 Anti-Myc protein G Dynabeads……..…………………………...…...20 2.4 Protein purification techniques.……………………..…………………………..20 2.4.1 Tandem affinity purification of ubiquitylated proteins from His8-biotinubiquitin expressing cells…………………………………...………….20 2.4.2 Purification of SILAC labeled ubiquitylated proteins using GST-S5a sepharose…………………………………...…………………………...22 2.4.3 Purification of FLAG-parkin and its interactors from SILAC cells expressing FLAG-parkin versus control……………………………….23 2.4.4 Formaldehyde cross-linking……………..…………………...……….24 2.4.5 Purification of cross-linked Myc-parkin………...…………………….24 2.5 Protein detection techniques.……………………..…………………………….25 2.5.1 Coomassie and silver staining………………………...………...…....25 2.5.2 Western blotting..………………………………...…………………….25 2.5.3 Immunofluorescence…………………………...……...………………26 2.6 Protein preparation for mass spectrometry.…………………………………...27 2.6.1 Trichloroacetic acid protein precipitation…………………..………...27 2.6.2 On-bead trypsin digestion of His8-biotin-ubiquitin conjugates……..27 2.6.3 In-gel digestion of GST-S5a bound proteins……………….…….…28 v  2.6.4 In-solution digestion of FLAG-parkin immunopreciptate…………...29 2.6.5 In-solution digestion of Myc-parkin immunoprecipitate………...…..29 2.7 Liquid chromatography and tandem mass spectrometry…………………….29 2.8 Analysis of mass spectrometry data……………………………………………30 2.8.1 Ingenuity pathway analysis……………………………...…………….32 CHAPTER 3. RESULTS.……………………..……………………..………………………..33 3.1 Usage of His8-biotin-ubiquitin as a tag based approach for isolating ubiquitin conjugates…………………………………………………………………………34 3.2 Identification of ubiquitylated proteins using the HBU system in neuronal cells…………………………………………………………………………….…..37 3.3 Preparation of an alternative ubiquitin conjugate purification strategy using S5a………………………………………………………………………………....40 3.4 Quantitative proteomic analysis of S5a affinity purified proteins…………….42 3.5 Direct immunoprecipitation of parkin binding proteins..………………………46 3.6 Capturing parkin interactors with cross-linking prior to immunoprecipitation52 3.7 Quantitative proteomic analysis of parkin interactors upon mitochondrial stress………………………………………………………………………………55 CHAPTER 4. DISCUSSION.…………………….…………………………………………...62 4.1 Ubiquitin purification methods for mammalian cells………………...…………63 4.2 Searching for parkin substrates by direct immunoprecipitation..……………66 4.3 Concluding remarks………………………………………………………………70 CHAPTER 5. REFERENCES………………………………………………………………...72 APPENDIX………………………………………………………………………………..……84  vi  LIST OF TABLES Table 1.1  Genes affected in PD…………………………………………………………...5  Table 2.1  Variable modification settings for Mascot searches………………………..31  Table 3.1  HBU enriched proteins – number of unique peptides……………………...39  Table 3.2  GST-S5a SILAC ratios of significantly enriched proteins………………….45  Table 3.3  Enriched protein ratios from FLAG-parkin pull-down..…………………….52  Table 3.4  Enriched proteins from CCCP treated FLAG-parkin lysate……………….59  vii  LIST OF FIGURES Figure 1.1  Clinical parkin mutations………………………………………………………..6  Figure 1.2  The ubiquitin-proteasome system….……………………………………….....7  Figure 1.3  Hypothesis: parkin-mediated mitophagy…………………………………….11  Figure 2.1  Tandem affinity purification of His8-biotin-ubiquitin……………..………….22  Figure 3.1  Tandem affinity purification of His8-biotin-ubiquitin conjugates….………..36  Figure 3.2  Preparation of GST-S5a and lysate pull-down.…………………………….41  Figure 3.3  GST-S5a SILAC enrichment graph…………..……………………………...44  Figure 3.4  Purification of FLAG-parkin interactors……………………………………...47  Figure 3.5  Quantitative mass spectrometry to determine parkin interactors.………..49  Figure 3.6  FLAG-parkin SILAC enrichment graph…………………………………...…50  Figure 3.7  Relative intensity versus m/z of parkin and secretogranin-2…………...…51  Figure 3.8  Purification of Myc-parkin interactors cross-linked lysate……..…………..54  Figure 3.9  Parkin recruitment to the mitochondria upon CCCP treatment..………….56  Figure 3.10 FLAG-parkin CCCP SILAC enrichment graph……………………………...57 Figure 3.11 Relative intensity versus m/z showing myosin-IX and myosin regulatory light chain peptides…………………………………………………………….58 Figure 3.12 Parkin solubility is reduced upon CCCP treatment………..…..…………..60 Figure 3.13 Cytochalasin D hampers parkin localization to mitochondria……………..61 Figure 4.1  Myosin IX domains………………………………...…………………………..69  viii  LIST OF ABBREVIATIONS  ACN  Acetonitrile  Arg  Arginine  ARJP  Autosomal recessive juvenile Parkinsonism  Asn  Asparagine  ATP  Adenosine triphosphate  BSA  Bovine serum albumin  CCCP  Carbonyl cyanide m-chlorophenyl hydrazone  CDC  Cell-division cycle  cDNA  complementary DNA  Da  Dalton  DC  Detergent compatible  DCM  Dilated cardiomyopathy  ddH2O  double distilled nanopure water  DMEM  Dulbecco’s Modified Eagle’s Media  DMSO  Dimethyl sulphoxide  DNA  Deoxyribonucleic acid  EDTA  Ethylenediaminetetraacetic acid  EGF  Epidermal growth factor  EGFR  Epidermal growth factor receptor  EndoLysC  Endoproteinase Lys-C  ER  Endoplasmic reticulum  ESCRT  endosomal sorting complex for transport  ESI  Electrospray ionization  ETC  Electron transport chain  FA  Formic acid  FBS  Fetal bovine serum  G418  Geneticin  GDP  Guanosine diphosphate ix  Gln  Glutamine  GlyGly  Glycine-glycine  GST  Glutathione S-transferase  GTP  Guanosine triphosphate  HB  His8-biotin  HBS  HEPES buffered saline  HBU  His8-biotin-ubiquitin  HCl  Hydrochloric acid  HDAC6  Histone deacetylase 6  His  Histidine  HPLC  High performance liquid chromatography  HRP  Horseradish peroxidase  IgG  Immunoglobulin G  IMAC  Immobilized metal affinity chromatography  IPTG  Isopropyl β-D-1-thiogalactopyranoside  KOAc  Potassium acetate  koff  Dissociation constant  LC  Liquid chromatography  LTQ  Linear trapping quadrupole  Lys  Lysine  MAIF  Mitochondrial apoptosis inducing factor  MG132  N-(benzyloxycarbonyl)leucinylleucinylleucinal  MRM  Multiple reaction monitoring  MS  Mass spectrometry  MS/MS  Tandem mass spectrometry  MW  Molecular weight  MWCO  Molecular weight cutoff  m/z  mass over charge ratio  OD  Optical density  Pael-R  Parkin-associated endothelin-like receptor  PAGE  Polyacrylamide gel electrophoresis x  PBS  Phosphate buffered saline  PCNA  Proliferating cell nuclear antigen  PCR  Polymerase chain reaction  PD  Parkinson’s disease  PFA  Paraformaldehyde  PINK1  phosphatase and tensin homolog-induced putative kinase 1  PMSF  Phenylmethylsulphonyl fluoride  ppm  parts per million  Q-TOF  Quadrupole Time-of-Flight  RING  Really interesting new gene  RNA  Ribonucleic acid  RT  Room temperature  SDS  Sodium dodecyl sulphate  SCX  Strong cation exchange  SILAC  Stable isotope labeling with amino acids in cell culture  siRNA  Small interfering ribonucleic acid  STAGE  STop and Go Extraction  TBS  Tris buffered saline  TCA  Trichloroacetic acid  TFA  Trifluoroacetic acid  TCEP  Tris(2-carboxyethyl)phosphine  Th  Thomson  TS  Tween-salt  UBA  Ubiquitin associated  UIM  Ubiquitin interacting motif  UPS  Ubiquitin proteasome system  WT  Wild-type  YFP  Yellow fluorescent protein  xi  ACKNOWLEDGEMENTS  I would like to my supervisor Dr. Thibault Mayor for his ideas and encouragement over these last couple of years and for giving me the opportunity to work on this exciting project. I would like to acknowledge my supervisory committee, comprised of Dr. Juergen Kast and Dr. Michel Roberge, for their time and helpful advice. I would also like to acknowledge Dr. Leonard Foster and his lab personnel for helping me during my research especially Dr. Nikolay Stoynov for his technical guidance in mass spectrometry. As for the entire Mayor Lab, I could not have imagined a better working and supportive environment. It was truly an enjoyable experience to be in the company of such wonderful people. Finally, I would like to thank my family for their support and confidence.  xii  CHAPTER 1 INTRODUCTION  1  1.1 The ubiquitin system Ubiquitylation is a major post-translational modification in the eukaryotic cell on par with phosphorylation1. Ubiquitin is a highly conserved 76-residue polypeptide that can be conjugated to target proteins via the ubiquitin C-terminal glycine carboxyl group2. Lysine side chains are the most common target sites within substrate proteins resulting in an amide (or isopeptide) bond between ubiquitin and substrate. A cascade of reactions is necessary to attach ubiquitin to proteins. First, the E1-activating enzyme uses ATP to form a high energy thioester bond with ubiquitin in which ubiquitin is then passed to the active cysteine of an E2-conjugating enzyme. Finally, the E3-ligase enzyme transfers ubiquitin from the E2 to specific substrates3. There are about six hundred E3 ubiquitin ligases in humans, reflecting how vast the ubiquitin system is3. Proteins can either undergo single or multiple mono-ubiquitylation or poly-ubiquitylation, in which a distal ubiquitin’s C-terminus is conjugated to one of seven lysine residues of ubiquitin, forming either a Lys-6, 11, 27, 29, 33, 48, or 63 isopeptide bond3. The ubiquitin chain may extend up to several ubiquitin molecules. Ubiquitin C-terminal hydrolases facilitate the reverse reaction of de-ubiquitylating substrates by cleaving after the last glycine residue of ubiquitin3. Different types of ubiquitylation reactions can occur resulting in diverse functions including membrane protein trafficking, chromatin dynamics, DNA repair and proteasomal degradation3. For instance, multiple mono-ubiquitylation of membrane proteins results in endocytosis by the endosomal sorting complex for transport (ESCRT) machinery into lysosomes for destruction, an important function for proper cell signaling and metabolism5. Ubiquitin is also involved in gene activation in the case of histone H2A  2  and H2B regulation of chromatin6-8. Mono-ubiquitylation and poly-ubiquitylation of proliferating cell nuclear antigen (PCNA) facilitates translesion synthesis and error-free DNA-damage-tolerance pathway activation respectively, processes which are essential for DNA repair9. A major function of ubiquitin is to target proteins for proteasomal degradation. The ubiquitin-proteasome system (UPS) involves substrates polyubiquitylated through Lys-48, the most common type of ubiquitin linkage, although recently mono-ubiquitylated substrates as well as Lys-11 and Lys-29 poly-ubiquitylation were also discovered as a target for proteasomal degradation10. Shuttling proteins including CDC48/p97 in some cases then transport the poly-ubiquitylated substrates to the 26S proteasome, a large protein complex. After binding, the substrates are deubiquitylated and unfolded prior to their entry into the catalytic core where they are cleaved into peptides and finally by cytosolic peptidases into amino acids11. The UPS is vital for eliminating misfolded proteins and a mechanism for protecting cells from their toxic effects and also recycling amino acids. Proteasome-dependent degradation clears misfolded proteins from the cytosol, nucleus and endoplasmic reticulum (ER)12. Besides functioning in protein quality control, the UPS also directly regulates cell cycle progression and apoptosis; for instance, ubiquitin ligase MDM2 targets p53, implicating the UPS in cancer progression13.  1.2 The UPS in Parkinson’s disease The presence of misfolded and aggregated proteins and inclusion bodies containing ubiquitin is a hallmark of many neurodegenerative diseases such as Alzheimer, Parkinson’s and Huntington14-15. Understanding the UPS is therefore key for  3  comprehending the molecular mechanisms underlying these diseases. Parkinson’s Disease (PD) is a debilitating condition characterized by resting tremor, rigidity and other features such as postural and autonomic instability16. Degeneration of dopaminergic neurons in the substantia nigra of the midbrain, in addition to other catecholamine and serotonin neurons in the brainstem are underlying causes of the disease17. Another hallmark of PD patients is the formation of Lewy bodies, which are intraneuronal inclusions enriched with ubiquitin, located in the cell body of surviving neurons18. One well accepted theory is that inclusion bodies are a cellular defense mechanism to sequester toxic misfolded proteins into single insoluble states, in conditions where the proteasome may be overwhelmed19. Inheriting forms of PD have been linked to mutations in three genes that are related to the UPS (Table 1.1): alpha-synuclein, ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) and parkin15, 20. Over-expression of alpha-synuclein, especially mutant forms, inhibits the UPS21. This effect is even more significant as triplication of the wild-type alpha-synuclein gene has been found to cause autosomal dominant PD22. An interesting proposed mechanism is that upon alpha-synuclein binding to the 19S proteasome complex, other substrates are not degraded due to hinderance by alphasynuclein docking23, 24.  4  Table 1.1 Genes affected in PD. Figure adapted from reference 16.  Another significant gene associated with inherited forms of PD is PARK2, coding for parkin (Table 1.1)25. Mutations in PARK2 are responsible for nearly 50% of autosomalrecessive juvenile-onset parkinsonism (ARJP)26, in which the onset of PD symptoms occur prior to the age of forty, as well as 10-15% of sporadic early onset PD. Parkin is an E3 ubiquitin ligase belonging to the RING family27. It contains three RING domains as well as an ubiquitin-like domain that is both structurally and sequence related to ubiquitin. Mutations causing ARJP include stop mutations, truncations and deletions that inactivate both alleles of PARK2 (Figure 1.1)28. Most of the mutations associated with PD impair the ligase activity of parkin. Parkin is the first ubiquitin ligase shown to bind eight Zn+2 ions through conserved cysteine-rich clusters which are essential for structural maintenance, an observation that rationalizes cysteine-based ARJP mutations found throughout parkin29. A simple model for how loss-of-function mutations in parkin could lead to PD suggests that the accumulation of one or more parkin substrates results in selective neuronal cell death (Figure 1.2)30. Over-expression of parkin can rescue cultured primary neuronal cells against toxicity due to expression of mutant alpha-synuclein, a substrate of parkin,  5  suggesting loss of E3 ligase activity is an important contributor towards ARJP etiology31. S-nitrosylation can also reduce parkin function suggesting oxidative modification of parkin as a cause for sporadic PD32. Homozygous loss-of-function parkin mutations are associated with a lack of Lewy bodies postulating that parkin may be involved in Lewy body formation to protect the cell from misfolded proteins33. This model is further strengthened by studies showing parkin poly-ubiquitylates misfolded DJ-1 via Lys-63 chains upon proteasomal inhibition resulting in trafficking along microtubules with the histone deacetylase 6 (HDAC6) adaptor protein and delivery of mutant DJ-1 to inclusion bodies34. e  Figure 1.1 Clinical parkin mutations. The grey and black circled mutations result in inactivation of parkin ligase activity in-vitro. Figure adapted from reference 142.  6  parkin
E3
  Figure 1.2 The ubiquitin-proteasome system. Parkin functions as an E3 ubiquitin ligase and conjugates ubiquitin to specific substrate proteins. Figure adapted from reference 143.  Several other parkin substrates satisfying the criteria of an authentic substrate have been identified including parkin-associated endothelin-like receptor (Pael-R), an ER membrane protein (Appendix, Table 1)35. Parkin is also involved in the trafficking regulation of the epidermal growth factor receptor (EGFR) through mono-ubiquitylation of the epidermal growth factor receptor substrate 15 (Eps15), thus delaying EGFR endocytosis upon EGF binding and prolonging phosphoinositide-3-kinase protein kinase B (PI3K-Akt) signaling through the activated EGFR leading to increased neuronal cell survival36. 7  Parkin substrates do not have a common recognition motif so one possibility is that parkin acts globally on misfolded proteins. For example, parkin has been shown to directly ubiquitylate expanded poly-glutamine proteins in vitro37 and associate with carboxyl terminus of Hsp70-interacting protein (CHIP), an E3 that targets misfolded proteins in a chaperone dependent manner30, 38. It will be important to determine the key substrate(s) of parkin contributing to PD upon parkin loss-of-function and how ubiquitylation may affect the substrates’ activity.  1.3 Parkin and the mitochondrial connection Mitochondrial dysfunction has also been shown to associate with PD – a deficiency in mitochondrial respiratory electron transport chain (ETC) nicotinamide adenine dinucleotide dehydrogenase (NADH) is consistently found in PD patients39. Alterations in antioxidant and oxidized targets have been reported in PD linking oxidative stress with the disease40. This observation further links mitochondrial dysfunction to PD since the mitochondrial ETC is a source of reactive oxygen species. However, the strongest evidence of a role for mitochondria in PD is emerging from the familial PD genes PARK2 and PINK1. Studies in model organisms lacking parkin suggest an important role for the protein in maintenance of mitochondrial function and integrity41, 42. Parkin loss-of-function mutants in Drosophila display increased sensitivity to oxygen radical stress, dopaminergic neuron loss and degneration of indirect flight muscles due to swollen, disordered mitochondria and fragmented cristae43, 44. Functional parkin is required for proper mitochondrial organization and morphology throughout spermatid development in Drosophila43, 45.  8  Narenda et al. conducted an elegant study in 2008 and found parkin targets dysfunctional mitochondria for autophagic degradation46. They depolarized the mitochondria with the protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and observed the recruitment of yellow fluorescent protein (YFP) tagged parkin to the mitochondria in several mammalian cell types resulting in mitochondrial fission and mitophagy. This study suggests parkin is an important component of mitochondria quality control. The phosphatase and tensin homolog (PTEN)-induced putative kinase 1, PINK1, is a nuclear-expressed mitochondria-targeted kinase, in which mutations are associated with autosomal recessive PD47. Mitochondrial respiration is selectively impaired in the striatum of PINK1 knock-out mice48. Drosophila expressing loss-of-function PINK1 or were PINK1 knock-out exhibited increased stress susceptibility, decreased ATP levels and mitochondrial morphological defects49-52. In all situations, parkin over-expression completely rescued the consequences of PINK1 deficiency whereas PINK1 overexpression did not rescue the phenotype of PARK2 knockout Drosophila, and double knockouts had the same level of defect. These studies point to both proteins being involved in the same pathway to regulate mitochondria function and stability in which parkin is downstream of PINK1. Since parkin acts downstream, PINK1 may recruit parkin to the mitochondria since parkin is largely cytosolic53. One line of evidence suggesting this mechanism is in neuroblastoma cells; over-expression of parkin alone resulted in cytosolic localization, but a co-expression with PINK1 resulted in parkin translocation to mitochondria, dependent on an active PINK kinase activity52 (PINK1 kinase domain faces the cytoplasm 54 ). There is also evidence of a direct 9  parkin-PINK1 interaction55. The model of PINK1 serving as a sensor of mitochondrial stress is strengthened by the fact that two proposed PINK1 substrates are also involved in quality control: the chaperone Trap1/Hsp7556 and the serine protease HtrA2/Omi57. Activation of Trap1/Hsp75, and HtrA2/Omi by PINK1 phosphorylation mediates degradation of unfolded or oxidized intermembrane space proteins, and refolding of damaged proteins and reduction of mitochondrial reactive oxygen species (ROS). A current reasonable model proposed by McBride58 is PINK1 initiates a signaling response through phosphorylation upon mitochondrial stress resulting in the translocation of parkin to damaged mitochondria, where PINK1 and parkin promote fission of damaged mitochondria59 (Figure 1.3). Parkin then recruits the autophagy machinery resulting in the engulfment of damaged mitochondria into autophagosomes and fusion with lysosomes. An accumulation of dysfunctional mitochondria may underlie the pathogenesis of PD upon a mutation in either gene. Ubiquitin ligase activity has been implicated in mitochondrial dynamics such as fission, fusion and trafficking60-62. This suggests the possibility of parkin inducing similar regulation of mitochondrial dynamics after translocation to the mitochondria. It will be pertinent to determine if parkin’s ubiquitylation activity is required for the mitophagy pathway and what these mitochondrial targets are, and to determine the potential function of ubiquitylation in targeting damaged mitochondrial fragments to LC3-autophagosomes. Identification of other proteins potentially facilitating the mitophagy pathway through parkin interactions will also be an important goal.  10  parkin recruitment and cleavage of damaged mitochondria by Drp1 mitochondria engulfment into pre-autophagosome  fusion into lysosomes for degradation  Figure 1.3 Hypothesis: parkin-mediated mitophagy. Parkin localizes to damaged mitochondria and promotes their autophagy suggesting a potential link between two genes implicated in PD, PARK2 and PINK1, a mitochondrial kinase which may serve as a sensor. Drp1 is a mitochondrial fission enzyme, ub is ubiquitin and LC3 is an autophagy marker. Figure adapted from reference 58.  1.4 Methods for the purification of ubiquitylated proteins There are likely several thousand ubiquitylated proteins in vivo undergoing temporal and spatial regulation, hence proteomics based methods are valuable to identify and characterize post-translationally modified proteins with ubiquitin63, 64. The turnover rate and labile conjugation (reversed by de-ubiquitylating enzymes) prevent the preservation of a high steady-state level of ubiquitin conjugates, which hindered initial studies63. The first breakthrough was achieved with peptide tags fused to ubiquitin such as hemagglutinin (HA), Myc and FLAG that are recognized by antibodies enabling affinity resin purification65. Biotin and poly-histidine tags have also been used66. Poly-histidinetagged ubiquitin can be expressed in eukaryotic cells and enriched by immobilized metal affinity chromatography (IMAC) under denaturing conditions on nickel beads. The 11  advantage of purification under a stringent environment is reduction of non-specific binding to nickel resins or to ubiquitin bound on the resin67.  1.4.1 Purification of ubiquitylated proteins from yeast Most global strategies have been applied to the yeast system68-71, but some studies have demonstrated feasibility in mammalian systems72-76. One prominent study conducted by Peng et al.68 in 2003 involved knocking out all four ubiquitin genes in yeast with reintroduction of His6-ubiquitin as the only ubiquitin source. 110 ubiquitylation sites on 72 different proteins were detected since the isopeptide bond between ubiquitin and the Lys on the substrate or another conjugated ubiquitin prevents tryptic digestion leaving a di-Gly motif. This study was the first to identify all seven Lys of ubiquitin participating in chain linkages. Tagwerker et al. 68 described a tandem affinity tag composed of His6-ubiquitin and a linker region followed by a biotinylation tag (biotinylated in eukaryotic cells) in vivo enabling tandem IMAC, streptavidin purification. 258 proteins involved in metabolism, translation and proteolysis were identified with high confidence in their study.  1.4.2 Purification of ubiquitylated proteins from mammalian cells Gururaja et al. 72 established a new approach using in-vitro ubiquitylation with HeLa cell extract after adding ATP along with a tagged ubiquitin. They were able to identify 22 proteasome subunits, 18 ubiquitylating enzymes, 4 ubiquitin domain proteins and 36 proteins associated with redox processes, endocytosis/vesicle trafficking, cytoskeleton, DNA damage/repair, calcium binding and mRNA splicing. Vasilescu et al.75 were able to 12  develop a novel technique using ubiquitin antibodies coupled to protein G-agarose in order to enrich for ubiquitylated proteins from MCF-7 breast cancer cells. Over 70 proteins were identified in this screen including E3 ligases making this technique a means for analyzing endogenous ubiquitylated proteins without relying on overexpressed or tagged ubiquitin.  The 26S proteasome contains a 50 kDa integral subunit called S5a, which is capable of binding ubiquitin in chains of four or more77. Mammalian S5a contains two independent poly-ubiquitin binding sites of about thirty residues each, termed ubiquitin interacting motif (UIM), relying on hydrophobic interactions78. S5a was exploited to serve as an affinity enrichment tool for poly-ubiquitin conjugates79. Weekes et al. used S5a-affinity chromatography to purify and identify hyper-ubiquitylated proteins in dilated cardiomyopathy (DCM) and observed elevated ubiquitylation levels in explanted hearts with DCM80. 27 proteins in the S5a bound fractions were identified by two-dimensional gel electrophoresis only from DCM hearts.  1.5 Project Aim: Identification of novel parkin E3 ubiquitin ligase substrates and interacting proteins at the basal level and upon mitochondrial stress. Several parkin targets and interacting proteins have been discovered (Appendix, Table 1); however, the mystery of critical parkin targets underlying PD has yet to be understood well. This project will focus on deciphering novel parkin interactors using a proteomics approach. More specifically, we will seek to identify proteins interacting with parkin upon mitochondrial stress.  13  CHAPTER 2 MATERIALS AND METHODS  14  MATERIALS AND METHODS All chemicals were from Sigma unless otherwise stated.  2.1 Mammalian expression construct cloning All DNA purification products were purchased from Qiagen and procedures carried out as described by the manufacturer. Oligonucleotide primers were from Integrated DNA Technologies.  2.1.1 His8-biotin-ubiquitin The His8-biotin sequence tag was generated by PCR with the forward primer 5’TTGGATCCACCATGGGACACCACCATCACCATCACCATCACCGG-3’ and reverse primer 5’-TTGAATTCTTAACCACCTCTTAGTCTTAAGAC-3’ against a His6-biotinubiquitin Ylp yeast expression vector (ubiquitin from S. cerevisiae) (kind gift from Dr. Peter Kaiser, UC Irvine). PCR products were purified with the PCR purification Kit. The amplicon and pcDNA3 mammalian expression vector (kind gift from Dr. Leonard Foster, UBC) were digested with BamHI, EcoRI (New England Biolabs). Cut vector was treated with calf-intestinal phosphatase (NEB) and cut amplicon and vector were gel purified using the Gel Extraction Kit and ligated with Quick Ligase (NEB) to generate a sequence in pcDNA3. XL-1 Blue competent E. coli were transformed with the ligated DNA and plasmid DNA was isolated with the QIAprep Spin Miniprep Kit. To generate a human ubiquitin sequence, 5’-TATT GCTAGCGGCGGCGGCGGCGGCATGCAGATCT TCGTCAAG -3’ and reverse primer 5’-TTGCTAGCTAACCACCTCTTAGTCTTAAGAC3’ was used in a PCR with ubiquitin-D77 (ubiquitin from Homo sapiens) pRSET and 15  amplicon was digested with NheI. His6-biotin-ubiquitin pcDNA3 was digested with XbaI (compatible cohesive ends) and the fragments processed as above to generate a construct expressing His8-biotin-ubiquitin with a human ubiquitin sequence and a five glycine residue linker between the biotin and ubiquitin. Plasmid transfection grade DNA was obtained using the QIAprep Spin Midiprep Kit.  2.1.2 His8-ubiquitin Yeast or human ubiquitin was amplified to generate a His8-ubiquitin controlled under a CMV promoter. Cloning procedures were identical to Chapter 2.1.1 except the forward primer used was 5’-TTGGATCCACCATGGGAGGTAGTCATCATCACCATCATCACCA TCATGGTGGTC AGATTTTCGTCAAGACTTTG-3’ and reverse primer was 5’-TTGAAT TCACCACCTCTTAGCCTTAGCAC-3’.  2.1.3 Myc-parkin Forward primer 5’-TTGAATTCTGATAGTGTTTGTCAGGTTC-3’ and reverse primer 5’TTGCGGCCGCTACACGTCAAACCAGTG-3’ was used in a PCR with FLAG-parkin pcDNA3 (parkin from R. norvegicus; kind gift from Dr. Edward Fon, McGill). Amplicon and N-Myc pcDNA3.1 (kind gift from Juergen Kast, UBC) were digested with EcoRI, NotI and processed as described in Chapter 2.1.1 His8-biotin-ubiquitin to obtain Mycparkin pcDNA3.1.  2.2 Cell culture techniques All cell culture materials were from Gibco unless otherwise stated. 16  2.2.1 Cell maintenance Human dopaminergic SH-SY5Y neuroblastoma were cultured in DMEM/F12 media with 10% heat-inactivated FBS and 1% penicillin/streptomycin. Cells were maintained at 37ºC in a saturated humidity atmosphere containing 95% air and 5% CO2. Cells were passaged by aspirating media, washing once with PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.4), incubating cells with 1ml 0.25% trypsin/EDTA (10cm dish) for 2 min at 37ºC, adding 3ml of media to neutralize trypsin, pipet mixing cells, and diluting 1/20 into fresh media. Cells were frozen by trypsinization, centrifugation at 4ºC 1000g 3 min followed by resuspension in 4ºC 90% FBS/10% DMSO solution at 1x106 cells/ml, and distribution into cryogenic tubes (Corning). Tubes were placed into the Mr. Frosty® container (Nalgene) and the container was then placed at -80ºC overnight. The next day, tubes were placed into a liquid nitrogen storage tank (Fisher Scientific).  2.2.2 DNA transfection Cells were seeded at 40-50% confluency on a 10cm dish (BD Biosciences) one day before transfection. Media was changed three hours before transfection and a solution of 20µg plasmid DNA, 1xHBS, and 1xCaCl2 at pH 7.10 (formula described by Kingston et al.81) was added to the cells for 16-20 hours. Cells were washed once with PBS and transfection media was replaced with new media and cells incubated for another 24-48 hours before further experimentation.  17  2.2.3 Generation of stable cell lines Cells were transfected in 10cm plates with the FLAG-parkin and Myc-parkin constructs using calcium phosphate transfection as described above. 72 hours after transfection, cells were trypsinized and replated in serial dilutions of 1:2, 1:20, 1:200, 1:2000 in media with 500µg/ml G418 selection drug for 14 days with media exchange every 2 days containing 500µg/ml G418. Media was removed and cells were washed with PBS and 20x diluted 0.25% trypsin/EDTA in PBS was layered over plates in which cell colony diameters reached about 1mm with good spacing between each colony. Cell colonies were picked with a P1000 pipet tip and aspirated into a labeled well of a 24 multi-well dish with medium. Clones were grown to 90% confluency and then expanded to 10cm dishes with 500µg/ml G418. Clones were finally collected to assess parkin expression by western blotting and frozen stocks made according to Chapter 2.2.1. Clones with sufficient FLAG-parkin and Myc-parkin expression were further assessed by immunofluorescence to evaluate expression purity.  2.2.4 SILAC Two cell populations were grown for six or more doublings in either SILAC DMEM/F12 media (Thermo Fisher) supplemented with 45.6 µg/ml 1H4-Lys and 73.8 µg/ml 12C6-Arg (light amino acids) or 45.6 µg/ml 2H4-Lys and 73.8 µg/ml 13C6-Arg (heavy amino acids) (Cambridge isotopes) respectively. Cells were initially seeded at 1/20 confluency on a 10cm dish, grown for one week and then expanded to at least 4x15cm dishes for a doubling of at least six times. All SILAC media was supplemented with 10% heat-  18  inactivated dialyzed FBS (Gibco) and 1% penicillin/streptomycin. After 6 or more doublings, cells were fully labeled and ready for further experimentation.  2.3 Preparation of biochemical purification reagents 2.3.1 GST-S5a sepharose 500 ml of BL-21 E. coli carrying the pGEX-5X-1-S5a sequence were grown to OD600 0.8 at 30ºC and induced with 0.8mM IPTG for 4 hours and subsequently washed with icecold TBS 3x100 ml and pelleted by centrifugation at 4ºC 5,000 rpm in a JA-10 rotor (Beckmann). Cells were lysed on ice with buffer containing 50mM sodium phosphate pH 8.0, 300mM NaCl, 0.5% Triton X-100, 1mM EDTA, 1mM PMSF and 1xprotease inhibitor cocktail (Roche). Lysate was then incubated with 5mM MgCl2 and 5µg/ml DNAse (Boehringer Mannheim) and sonicated (Misonix) with setting 3, 0.5sec ON/OFF pulses for 3x20sec with 1 min intervals on ice. Unbroken cells and debris were pelleted by centrifugation at 15,000rpm at 4ºC in a JA-20 rotor (Beckmann) and the supernatant incubated with 2ml glutathione sepharose resin (GE Healthcare) for 1 hour at 4ºC on a rotating platform (Fisher), washed with 3x10 resin volumes of lysis buffer and bound GST-S5a eluted with 1xresin volume of elution buffer (20mM glutathione in 50mM Tris pH 8.0). Glutathione sepharose resin was washed several times with lysis buffer and the binding/washing/elution steps were repeated one more time with the lysate supernatant flow-through from the first binding step to capture remaining GST-S5a. GST-S5a elution was dialyzed at RT for 1 hour three times against HEPES pH 7.5 in a 12-14,000 MWCO membrane (Spectrum) to remove free glutathione. GST-S5a was then coupled to Affigel 15 activated sepharose resin (Bio-Rad). Affigel was aliquoted 19  into a 10ml chromatography column (Bio-Rad) (1ml of Affigel used per 6mg of protein) and washed with ice-cold double distilled water (ddH2O). Dialyzed GST-S5a was added to Affigel and rotated at 4ºC for 4 hours. 100mM ethanolamine pH 8.0 final concentration, was added to the protein solution-Affigel mixture and incubated for 1 hour at RT to quench unreacted N-hydroxysuccinimide esters on the Affigel. GST-S5a coupled Affigel was washed with 3x10 resin volumes of HEPES pH 7.5 and stored with one resin volume of HEPES pH 7.5 with 0.02% NaN3 at 4ºC.  2.3.2 Anti-Myc protein G Dynabeads 1mg of 9E10 anti-Myc antibody (produced by BRC Antibody Facility, UBC) was added to 1ml of protein G Dynabeads (Invitrogen) and the coupling reaction was performed according to protein G Dynabeads manufacturer’s instructions.  2.4 Protein purification techniques 2.4.1 Tandem affinity purification of ubiquitylated proteins from His8-biotin-ubiquitin expressing cells The purification scheme is depicted in Figure 2.1. 2x107 cells were transfected with 35µg HBU pcDNA3 construct on 15cm diameter plates as described in Chapter 2.2. 41 hours after transfection, half the plates were treated with 20µM MG132 and the other half with DMSO for 7 hours. 48 hours after transfection, cells attached to plate were washed once with PBS, trypsinized and transferred into 15ml ice cold conical tubes (Sarstedt) and pelleted using a tabletop centrifuge at 1000g, 4ºC for 3 min (Eppendorf). Cells pellets were washed twice with ice cold PBS and lysed at RT with 2ml Buffer A 20  (8M urea, 300mM NaCl, 50mM sodium phosphate pH 8.0, 0.5% Nonidet P-40, 1mM PMSF, 10mM chloroacetamide), passaged ten times through a 27G1/2 needle attached to a 1ml syringe (Fisher Scientific) and centrifuged at 15000g at 20ºC for 30 min to clear the lysate. Protein concentrations in the two lysates were measured with the DC Protein Assay Kit (Bio-Rad) and Buffer A added to normalize total protein quantities and volume. Imidazole was added to the supernatant at 10mM and lysate was incubated overnight with MagneHis beads (Promega) at 12µl beads slurry/mg protein lysate. A magnetic rack was used to separate the magnetic beads from the liquid phase. Beads were washed with Buffer A twice and once with Buffer A containing 10mM imidazole, and bound proteins eluted with 2x120µl Buffer B (8M urea, 200mM NaCl, 50mM sodium phosphate, 2% SDS, 10mM EDTA, 100mM Tris, 500mM imidazole pH 8.0). Eluate was then incubated with Dynabeads MyOne Streptavidin (Invitrogen) overnight at 7 µl beads slurry per mg of initial protein lysate and then beads were washed twice with Buffer C (8M urea, 200mM NaCl, 2% SDS, 100mM Tris pH 8.0); once with Buffer C containing 100mM β-mercaptoethanol (to cleave E1, E2 and E3 enzymes from ubiquitin); twice with Buffer D (8M urea, 1.2M NaCl, 0.2% SDS, 100mM Tris, 10% ethanol, 10% isopropanol pH 8.0) to remove hydrophobically bound material; and three times with Buffer E (8M urea, 50mM HEPES).  21  Figure 2.1 Tandem affinity purification of His8-biotin-ubiquitin. Figure adapted from reference 23.  2.4.2 Purification of SILAC labeled ubiquitylated proteins using GST-S5a sepharose Two populations of light and heavy labeled cells (prepared as described in Chapter 2.2.4 were treated with DMSO and 20µM MG132 for 7 hours respectively (2x107 cells per condition). Cells were collected as described in Chapter 2.4.1 and lysed on ice, each with 2ml lysis buffer (50mM Tris pH 8.0, 150mM NaCl, 0.5% Triton X-100, 25mM chloroacetamide, 1mM 1,10-phenanthroline, 1mM PMSF and 1xprotease inhibitor cocktail mix), needle passaged and centrifuged at 16200g for 15 min at 4ºC in a microcentrifuge. Proteins concentrations were measured, normalized and cell lysates from the two cell populations were mixed together. Lysate supernatant was pre-cleared with 100µl ethanolamine quenched Affigel 15 sepharose (pre-washed with lysis buffer)  22  for 1 hour at 4ºC and then incubated with 50 µg GST-S5a sepharose per mg of lysate for 4 hours at 4ºC. Resin was washed with 3x10 resin volumes of lysis buffer and bound proteins eluted with twice with 1 resin volume of 8M urea, 50mM HEPES pH 8.0.  2.4.3 Purification of FLAG-parkin and its interactors from SILAC cells expressing FLAGparkin versus control Two populations of cells, in which only one stably expressed FLAG-parkin and the other did not were labeled with light and heavy amino acids respectively. Cells were collected on ice and lysed separately with 2ml of lysis buffer (50mM HEPES pH 7.5, 0.5% Triton X-100, 10% glycerol, 70 mM KOAc, 0.2mM EDTA, 5mM chloroacetamide, 1mM PMSF, and 1xprotease inhibitor cocktail mix). Lysates were passaged, centrifuged and protein concentrations normalized. 6µl of anti-FLAG M2 affinity resin were used per mg of protein lysate and the mixture was incubated at 4ºC for 4 hours on a rocking platform. M2 resin was pelleted by centrifugation at 5000g for 2 min after completed incubation and pooled together. Resin was washed sequentially with 10 resin volumes of lysis buffer, twice with 10 resin volumes of 50mM Tris pH 7.5, 150mM NaCl, 1% Tween (potentially releasing weak interacting proteins) and twice with 10 resin volumes of 50mM Tris pH 7.5, 500mM NaCl (also weak interactor elution). FLAG-parkin and more tightly associated proteins were eluted twice with 1 resin volume of sample buffer containing 50mM Tris pH 6.8, 2% SDS, 10% glycerol (strong interactor elution). For the CCCP experiment, two SILAC cell populations expressing FLAG-parkin were treated with DMSO or with 10µM CCCP for 3 hours prior to cell collection. All other procedures are identical to the non-CCCP SILAC experiment.  23  2.4.4 Formaldehyde cross-linking optimization Paraformaldehyde was dissolved in PBS at 0.00%, 0.20%, 0.40%, 0.60%, 0.80% with gentle heating. SH-SY5Y stably expressing Myc-parkin were grown on 6cm dishes to 90% confluency, washed with PBS and various concentrations of formaldehyde solution were added and plates rocked on a shaker (Fisher Scientific) for 10 min. Cross-linking reaction was quenched with addition of 1.25M glycine pH 7.4 to a final concentration of 125mM for a 5 min incubation period. Cells were washed twice with PBS, trypsinized, collected in ice-cold tubes and pelleted. Cells were lysed with Buffer F (50mM Tris pH 7.5, 150mM NaCl, 10% glycerol, 1% Nonidet P-40, 5mM EDTA, 1mM PMSF, 1xprotease inhibitors). Protein loading was normalized for SDS-PAGE and parkin western blotting to determine the optimum concentration of formaldehyde treatment.  2.4.5 Purification of cross-linked Myc-parkin Two cell populations (6x106 cells per population) where one stably expressed Mycparkin, were treated with 0.40% formaldehyde solution, the optimal determined concentration (Figure 3.8A). Cells were lysed with Buffer F and protein concentrations normalized. Lysates were added to pre-equilibrated 9E10-coupled Dynabeads at 50µl beads slurry per mg of protein and incubated for 2 hours at 4ºC. Beads were washed three times with 10 bead volumes of Buffer F and bound proteins eluted with 2 resin volumes of sample buffer (2% SDS, 50mM Tris pH 6.8, 10% glycerol). Formaldehyde cross-links were reversed by heating the protein mixture at 95ºC for 10 min. Parkin immunoblotting revealed much of the Myc-parkin eluted in the 0.5% SDS fraction  24  (Figure 3.8B), thus the material was precipitated using chloroform and methanol as previously described82 and processed further.  2.5 Protein detection techniques 2.5.1 Coomassie and silver staining Cell lysates prepared as described in Chapter 2.4 were reconstituted in 3x Laemmli sample buffer (150mM Tris pH 6.8, 6% SDS, 30% glycerol, 6% β-mercaptoethanol, bromophenol blue) and loaded on pre-cast 4-20% gradient polyacrylamide gels (Pierce). SDS-PAGE was performed at 150 volts for 45 min on a Novex electrophoresis device (Invitrogen, Bio-Rad). For coomassie staining, gels were incubated in 0.2% coomassie blue R350 solution with 40% methanol and destained with 40% methanol, 10% acetic acid solution for visualization of proteins in gels. For silver staining, gels were fixed with 30% ethanol, 10% acetic acid for 3x20 min and incubated in 0.1% sodium thiosulphate dissolved in solution A (30% ethanol, 10mM sodium acetate, pH 6 with acetic acid) for 1 hour followed by 3x20 min ddH2O washes. Gels were then incubated in 0.1% silver nitrate, 0.01% formaldehyde solution for 1 hour, washed and developed with 2.5% sodium carbonate 0.02% formaldehyde solution for 5 min and quenched with 1% acetic acid.  2.5.2 Western blotting After SDS-PAGE, proteins were transferred onto 0.5µm nitrocellulose membranes (BioRad) using the wet-transfer technique in Towbin buffer (25mM Tris, 192mM glycine, 25  20% methanol, pH 8.3) and a transfer device set at constant 0.50 ampere for 2 hours at 4ºC (Hoefer). For ubiquitin immunoblotting, membranes were microwaved in ddH2O for 15 min prior to blocking to unfold mono-ubiquitin. Membranes were blocked with blocking buffer (3% skim milk powder dissolved in TBS, 0.1% Tween-20) for 30 min followed by primary antibody incubation dissolved in blocking buffer (P4G7, ubiquitin antibody 1:1000 (Santa Cruz) or PRK8, parkin antibody 1:1000 (Santa Cruz)) for 1 hour at RT. Membranes were washed 2x5 min with blocking buffer and then incubated with Goat anti-mouse HRP 1:3000 (Bio-Rad) for 45 min. Membranes were washed 5 min successively with blocking buffer, blocking buffer without milk, and TBS followed by chemiluminescence substrate reagent incubation (Perkin Elmer) and development on Biomax film (Kodak). ExtrAvidin® peroxidase was used to detect biotin according to manufacturer’s instructions.  2.5.3 Immunofluorescence Cells were seeded at 25% confluency on coverslips pre-treated with 1M HCl (Fisher Scientific). Media was removed 48 hours later and cells were gently washed once with PBS before incubation with 3% PFA in PBS for 15 min at RT. Cells were permeabilized with 0.5% Triton X-100 for 2 min, PBS washed, and blocked with 1% BSA (Roche) in PBS for 10 min. PRK8 antibody was diluted in 3% BSA at 1:1000 and incubated for 45 min with coverslips followed by 3x PBS washes. Alexa Fluor 488 Goat anti-mouse secondary antibody (Invitrogen) diluted 1:2000 in 3% BSA was then added for 45min. Coverslips were mounted on glass slides (Fisher scientific) with ProLong Gold Antifade reagent (Invitrogen). For mitochondria staining, cells were incubated with 400nM 26  MitoTracker Orange CMTMRos (kind gift from Dr. Leonard Foster, UBC) for 45 min prior to PFA fixation. Slides were analyzed on a Carl Zeiss Observer.Z1 microscope with a Colibri 62HE filter set and Axiovision Rel. 4.7 software. Forty z-stack images separated by 0.2µm on the vertical axis were captured per image panel and inverse filter deconvolution was performed to increase image quality.  2.6 Protein preparation for mass spectrometry 2.6.1 Trichloroacetic acid protein precipitation Protein mixtures from the purified fractions described in Chapter 2.4.1, 2.4.5 were subjected to precipitation in order to remove detergents and concentrate proteins for mass spectrometry compatibility. TCA at a final concentration of 30% was added to protein mixtures and incubated overnight at 4ºC. Precipitants were pelleted by centrifugation at 4ºC for 15 min and washed twice with 100% ice cold acetone. Pellets were resuspended with 40µl 50mM HEPES pH 8.0 and 8M urea and the pH adjusted to 8.0.  2.6.2 On-bead trypsin digestion of His8-biotin-ubiquitin conjugates His8-biotin-ubiquitin conjugates bound to streptavidin beads following tandem affinity purification as described in Chapter 2.4.1 were resuspended in 125µl 8M urea, 50mM HEPES. Proteins were reduced with 3µM TCEP for 20 min and alkylated with 55mM chloroacetamide for 30 min successively in the dark at 1400rpm on a Thermomixer set at 25ºC (Eppendorf). 0.1µg of endo-LysC (Roche) was added and digestion proceeded for 3 hours at 37ºC with intermittent 5 sec ON/25 sec OFF on a Thermomixer. Protein 27  solution was diluted 4 fold with dropwise addition of 100mM Tris pH 8.5 and addition of 1mM CaCl2. 1.5µg sequencing grade trypsin (Roche) was then added and digestion proceeded similarly for 16 hours until digestion termination and peptide acidification with the addition of 10% FA. Residual peptides on the beads were extracted with 3x40 µl 0.1% FA, 25% ACN and the three extracts were pooled and concentrated with a vacuum centrifuge until ~20µl (2 hours) (Eppendorf). Peptides were purified and concentrated on a STAGE tip83-85 with one C18 column and eluted with 80% ACN, 0.5% acetic acid into 96 well plates; dried in vacuum concentrator; and resuspended in sample buffer containing 1% TFA, 0.5% acetic acid and 3% ACN.  2.6.3 In-gel digestion of GST-S5a bound proteins TCA precipitant derived from Chapter 2.4.2 was resuspended with 8M urea, 50mM HEPES pH 8.0 and reconstituted with 3xLaemmli buffer and loaded onto a 4-20% gradient polyacrylamide gel. Bromophenol blue marker dye was run for halfway down the gel (150 volts, 20 min). Gel bands were excised with a scapel corresponding to molecular weight ranges of >200, 100-200, 50-100, <50 kDa and pieces were further minced into approximately 1mm3 cubes to enable improved reagent exchange. Gel samples were subjected to in-gel digestion as previously described86 without gel staining to increase peptide identification87. Peptides from the four gel slices were purified and concentrated on a STAGE tip with one C18 column each and eluted with 80% ACN, 0.5% acetic acid into 96 well plates; dried in a vacuum concentrator (Eppendorf); and resuspended in sample buffer containing 1% TFA, 0.5% acetic acid and 3% ACN. 28  2.6.4 In-solution digestion of FLAG-parkin immunoprecipitate Weak and strong interactor TCA precipitants derived from Chapter 2.4.3 were resuspended with 40µl 8M urea, 50mM HEPES and pH adjusted to 8.0. Proteins were reduced, alkylated and digested as in Chapter 2.6.2, with the exception of endo-LysC. After 10% FA addition to quench the digestion, tryptic peptides were concentrated on STAGE tips containing a C18 column on top of an SCX column. Peptides were separated into 4 fractions with increasing concentrations of NH4OAc at 100mM, 350mM, 500mM and 1M to elute the peptides from the SCX column. Purified fractions were subjected to another C18 STAGE tip processing each and eluted with 80% ACN, 0.5% acetic acid into 96 well plates; dried in vacuum concentrator (Eppendorf); and resuspended in sample buffer containing 1% TFA, 0.5% acetic acid and 3% ACN.  2.6.5 In-solution digestion of Myc-parkin immunoprecipitant Chloroform-methanol precipitated proteins from Chapter 2.4.5 were processed as described in Chapter 2.6.2. After 10% FA addition to quench the digestion, tryptic peptides were purified and concentrated on STAGE tips containing a C18 column; eluted with 80% ACN, 0.5% acetic acid into 96 well plates; dried in vacuum concentrator (Eppendorf); and resuspended in sample buffer containing 1% TFA, 0.5% acetic acid and 3% ACN.  2.7 Liquid chromatography and tandem mass spectrometry Peptide samples were analyzed on an LTQ-Orbitrap (Thermo Electron). The LTQOrbitrap system was on-line coupled to Agilent 1100 Series nanoflow HPLC instruments 29  using nanospray ionization sources (Proxeon Biosystems) containing columns packed into 15cm long, 75µm inner diameter fused silica emitters (8µm diameter opening, pulled on a P-2000 laser puller from Sutter instruments) using 3µm diameter ReproSil Pur C18-AQ beads (Dr. Maisch). Buffer A (0.5% acetic acid) and Buffer B (0.5% acetic acid, 80% ACN). Gradients were run from 6% B to 30% B complemented with Buffer A over 54 min, then 30% to 80% B over the next 5 min, held at 80% B for 10 min, and then dropped to 6% B for another 10min to recondition the column. The HPLC system included Agilent 1100 series Degaser, Nano-flow pump, Autosampler and Thermostat. The termostat temperature was set at 6°C. The LTQ-Orbitrap was programmed to capture a full-range scan at 60,000 resolution from 350 to 1500 m/z in the Orbitrap and to simultaneously fragment the top five peptide ions in each cycle in the LTQ (minimum intensity 1000 counts, MS/MS starting after 20 min). Parent ions were then excluded from MS/MS for the next 30 sec. Singly charged ions were excluded since in ESI mode peptides usually carry multiple charges. An exclusion peptide list for anti-FLAG immunoglobulin and GST-S5a was programmed for anti-FLAG immunoprecipitated proteins and GST-S5a purified proteins respectively (Appendix, Table 2). The Orbitrap was continuously recalibrated using lock-mass function88.  2.8 Analysis of mass spectrometry data Raw MS spectra files were processed to Mascot generic format (mgf) using DTA Supercharge v.1.37 in order to correct spectrum file monoisotopic peak and charge states. Peak lists were searched against the International Protein Index Human 3.60 database (also Rat 3.60 database for parkin) compiled by the European Bioinformatics 30  Institute using Mascot. All searches included the following settings: trypsin/P cleavage specificity with up to two missed cleavages, cysteine carbamidomethyl fixed modification, ±20-ppm peptide tolerance, ±0.6-Da MS/MS tolerance, ESI-TRAP scoring scheme, up to two 13C, and peptide charges of +1, +2 and +3. Each sample had additional variable modifications (Table 2.1). Assigned peak lists from SCX fractions were pooled using Proteus software (Genologics). After comparing the number of identified peptides from the decoy database (protein sequences in database reversed) to the real database generated by Mascot for all samples, the minimum peptide threshold score for a false positive rate ≤1% was 25. MSQuant version 1.5 was used for parsing Mascot files and iterative mass recalibration. Peptides originating from keratin were manually removed from all data sets. Peptides originating from immunoglobulin chains were manually removed from FLAG-parkin immunoprecipitate and peptides from GST-S5a were removed from GST-S5a resin purified material.  Table 2.1 Mascot variable modification settings for pull-down mascot generic format files.  Mascot variable modification protein N-terminal acetylation Asn/Gln deamidation Met oxidation Lys-GlyGly isopeptide linkage Lys (2H4)-GlyGly Lys (2H4) Arg (13C6)  Immunoprecipitated Sample His8-biotin-ubiquitin/Myc-parkin S5a FLAG-parkin yes no yes yes no yes yes yes yes yes yes no no yes no no yes yes no yes yes  31  2.8.1 Ingenuity pathway analysis Protein SILAC ratio data was subjected to signaling pathway analysis with Ingenuity Pathway Analysis software v.8 (Ingenuity). The threshold for enrichment or deenrichment significance was set at a log2(heavy/light) absolute value = 0.57 for the S5a SILAC experiment.  32  CHAPTER 3 RESULTS  33  3.1 Use of His8-biotin-ubiquitin as a tag-based approach for isolating ubiquitin conjugates. We first sought to determine whether we could identify parkin substrates by enriching for ubiquitylated proteins. The rationale is parkin substrates should be purified to a greater extent from cells over-expressing parkin relative to cells with lower parkin expression due to increased parkin-mediated ubiquitylation. SH-SY5Y human neuroblastoma cells were chosen because they are brain cells with endogenous parkin expression. In addition, dopamine-producing cells are typically affected in Parkinson’s disease, making them relevant for studying PD etiology89. We chose the His6-biotinubiquitin tandem purification approach described by Tagwerker et al.69, because they identified the largest number of proteins by mass spectrometry at 258 compared to other studies (see Chapter 1.4). Another advantage of their method is that it enables purification under denaturing conditions resulting in lower background. The only caveat is the His6-biotin-ubiquitin system had only been developed in yeast when we initiated the project, and required some modification to be used in mammalian cells. We cloned a His8-biotin-ubiquitin, termed HBU, in the pcDNA3 mammalian expression vector. The construct contained a human ubiquitin sequence preceded by an octahistidine tag enabling a first step IMAC purification with urea and SDS to reduce nonspecific binding. The second tag is a 75 amino acid long sequence which is biotinylated in vivo by endogenous biotin ligase enabling a second affinity capture step with streptavidin beads90. The extension of His6 to His8 has no effect on ubiquitin conjugation (Mayor, T.; personal communication). Five glycine residues were inserted between the biotin sequence and ubiquitin to serve as a linker region. A control construct as a 34  byproduct of the cloning of HBU was His8-biotin, termed HB, in which a stop codon was in frame after the biotinylation sequence tag. After cellular transfection with constructs, free HBU was detectable with the P4G7 anti-ubiquitin antibody in lysates of cells transfected with HBU in contrast to HB transfected cells (Figure 3.1A). The expression level of free HBU relative to free ubiquitin was about a 2:3 ratio. We confirmed that HBU was conjugated to substrate by using horseradish peroxidase coupled to avidin to blot for biotin, albeit not with the same efficiency as wild-type ubiquitin (Figure 3.1B, compare ratio of mono-ubiquitin to poly-ubiquitin versus mono-HBU and poly-HBU in 3.1A). In contrast, the product of the control HB construct was not conjugated.  35  A  B  1 C  2  3  4  1  2  3  D  HBU ubiquitin Figure 3.1 Tandem affinity purification of His8-biotin-ubiquitin conjugates. Cells transfected with HBU or HB control were lysed and subjected to SDS-PAGE and immunoblotting for ubiquitin (A) and biotin (B) using a P4G7 anti-ubiquitin antibody and ExtrAvidin respectively. Marker positions in kDa are indicated. Aliquots at different stages of the tandem purification scheme were subjected to SDS-PAGE and immunoblotting with P4G7 (C) or silver staining (D). In C, the corresponding aliquots were assessed: lane 1 (1x) input, lane 2 (1x) Ni2+ resin unbound, lane 3 (3x) Ni2+ resin elution, lane 4 (3x) streptavidin beads unbound. In D, the corresponding aliquots were assessed: lane 1 (0.1x) input, lane 2 (10x) Ni2+ resin elution, lane 3 (10x) streptavidin beads unbound. There is no streptavidin elution lane because the interaction between biotin and streptavidin is nearly the strength of a covalent bond and no bound material was recovered (data not shown).  36  3.2 Identification of ubiquitylated proteins using the HBU system in neuronal cells. We next sought to purify HBU conjugates to assess whether ubiquitylated substrates and known parkin targets could be identified by MS using this approach. We performed a tandem purification to enrich for ubiquitylated proteins from cells transfected with HBU. Cells were lysed in 8M urea with SDS and incubated with Ni2+ beads. After elution, ubiquitylated proteins were further enriched using streptavidin beads. Stringent washes with detergent and alcohol were performed to remove non-specific hydrophobically bound proteins. The Ni2+ pull-down efficiency of ubiquitylated conjugates was about 20% (Figure 3.1C compare ubiquitin signal from elution to input lane). The depletion of ubiquitin signals in the streptavidin-unbound lane suggested that almost all of the HBU conjugates in the Ni2+ eluate were bound to the streptavidin beads (Figure 3.1, lane 4). This second purification step using streptavidin was important as revealed by the amount of non-specific proteins present in the streptavidin-unbound lane (Figure 3.1D, lane 3). Based on these encouraging results, we decided to identify by MS which proteins were purified by our method. We compared two samples (1x107 cells each) that were transfected with HBU and were either proteasome inhibited with MG132 or treated with the DMSO control solvent. Whether the HBU approach would be sufficient for the study of parkin ligase substrates depends on the number of proteins identified. Proteins were digested by sequencing grade trypsin directly on streptavidin beads and the whole peptide mixture was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) using an LTQ-Orbitrap as previously described91. Spectra were then  37  processed and analyzed by Mascot. We identified 20 proteins in the absence of proteasome inhibition and a larger number of proteins upon MG132 addition. We identified a total of 43 proteins by combining both samples with a minimum number of 2 peptides when the MS data list was searched with Mascot (Table 3.1). The top protein hit identified was ubiquitin suggesting the method is specific for enriching ubiquitin and its conjugates. The relatively low number of proteins identified compared to other studies69, 72-76 was not encouraging. In addition, most proteins were very abundant in the cells (e.g. ribosomes92) indicating the method was unlikely to pick low abundant proteins such as potential parkin substrates. In order to scale up, we generated a stable cell population expressing HBU by geneticin selection. We successfully established a stable population expressing HBU; however, the expression level was much lower compared to transiently transfected cells and purification of conjugated proteins was not efficient (data not shown). We also became aware that the HB tag was preventing ubiquitylation of some substrates (Deshaies, R.J.; personal communication) and therefore, we decided to test an alternative approach.  38  Table 3.1 HBU enriched proteins – number of unique peptides. A subset of the 41 proteins with a minimum of 2 peptides are shown. # beside the protein name represents a known parkin ubiquitin ligase substrate93.  Proteins identified from HBU pull-down in the presence of DMSO or MG132 ubiquitin ribosomal protein L19 ribosomal protein L3 tubulin α-1A chain# heat shock protein 71 heat shock protein 90 ribosomal protein L15 S5a proteasome receptor serine/threonine-protein kinase PRP4 actin ribosomal protein L13 sequestrome-1 putative RNA-binding protein Luc7-like 1 NEDD8 catenin beta-1 neutral amino acid transporter N-Myc proto-oncogene protein DNA-directed RNA polymerase II subunit RPB2 XLas-1 guanine nucleotide-binding protein hypoxia-inducible factor 1 alpha 14-3-3 zeta/delta protein ADP/ATP translocase 3-hydroxy-3-methylglutaryl-coenzyme A reductase DNA-damage-inducible transcript 4 protein tubulin β chain# histone H2A  unique peptides (DMSO) 7 2 2 3 3 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4  unique peptides (MG132) 10 6 4 4 4 4 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2  39  3.3 Preparation of an alternative ubiquitin conjugate purification strategy using S5a. We generated recombinant GST-S5a coupled to sepharose for S5a affinity chromatography as an alternative means to enrich for ubiquitin conjugates. S5a is a proteasome subunit that can bind to poly-ubiquitin and was previously used to enrich for ubiquitylated substrates79. GST-S5a was expressed in BL21 bacteria after IPTG induction (Figure 3.2B, lane 2). We performed a sequential pull-down until full depletion of recombinant GST-S5a (Figure 3.2A, lane 4). Pure GST-S5a was eluted from the glutathione sepharose resin (Figure 3.2A, lanes 5 to 8). We then dialyzed the GST-S5a solution to remove glutathione and successfully coupled the protein to activated sepharose resin with an efficiency of about 90% (Figure 3.2A, lanes 13 and 12; the flowthrough signal was less than the 25% input signal).  40  1 A  1  2  3  4  5  6  7  1 2 3  148  4  5 B  98 64 50 36 22 16 6  C GST-S5a MG132  input +  elution - + + +  250 160 110 80 60 50 40 30 20 15 10 3.5 1  2  3  4  Figure 3.2 Preparation of GST-S5a and lysate pull-down. Preparation of GST-S5a sepharose (A). Expression and purification of GST-S5a was assessed by SDS-PAGE followed by coomassie staining: Protein ladder in kDa is indicated on the left, lane 1 input, lane 2 first flow-through, lane 3 second flow-through, lane 4 and 5 first two elutions, lane 6 and 7 last two elutions. Coupling of GST-S5a: lane 9 input 1/2400, lane 10 input 0.75/2400, lane 11 input 0.50/2400, lane 12 input 0.25/2400, lane 13 affigel flow-through 1/2400. The arrow marks GST-S5a at 67 kDa. GST-S5a lysate pull-down was assessed by SDS-PAGE followed by silver staining (C). SH-SY5Y were treated with DMSO or MG132, lysed and subjected to pull-down with GST-S5a resin. Quenched Affigel 15 was used as resin to assess background binding in lane 3. Molecular weight marker in kDa are indicated on the left, lane 1 is lysate input, lanes 2-4 are purified protein fractions from the chromatography resin derived from cells treated as indicated.  41  3.4 Quantitative proteomic analysis of S5a affinity purified proteins We assessed the amount of coverage for the ubiquitin proteome after S5a purification was used to enrich for ubiquitin conjugates. We incubated cell lysate with the GST-S5a sepharose followed by high salt wash buffer (1 M NaCl), resulting in low background with the control resin (Figure 3.2B, lane 3). We then applied MG132 and noticed an overall increase of the bands associated to S5a. Ubiquitin immunoblotting revealed a pull-down efficiency of ubiquitin conjugates of about 50% (data not shown). Free ubiquitin was not enriched since S5a has poor affinity for monomeric ubiquitin. In order to determine whether this approach was suitable for ubiquitin proteome studies, we then performed stable isotope labeling with amino acids in cell culture94 (SILAC) to identify ubiquitylated protein accumulation upon proteasome inhibition. S5a had been previously used, but without a careful control79. Light and heavy labeled cells were treated with DMSO and MG132 respectively. After the enrichment with S5a, the protein mixture was separated by SDS-PAGE and four gel pieces corresponding to >200 kDa, 100-200 kDa, 50-100 kDa and <50 kDa were excised and subjected to in-gel trypsin digestion86 followed by LC-MS/MS. After pooling the four fractions using Proteus, we identified 310 proteins (false positive rate ≤1%) by Mascot. About 20% of the identified quantified by MSQuant were specifically enriched from MG132 treated cells (Figure 3.3, log2(heavy/light) ≥0.57; Table 3.2). We chose to use 0.57 as the cut-off for enrichment upon proteasomal inhibition because it is the point where ratios start to deviate higher than the linear trend relationship of log2ratio versus protein number as observed between proteins 75 and 290 (Figure 3.3). MCM3, an initiator of genome replication, was the last protein meeting the cut-off and it is a known proteasome substrate95. 42  Ubiquitin was enriched above the cut-off at a log2 ratio of 0.69. Proteins not enriched (log2(heavy/light) ~ 0) may also be ubiquitylated but not affected by proteasome inhibition. Some examples of known proteasome substrates enriched include proliferating cell nuclear antigen (PCNA)96, 97 and acetyl CoA-carboxylase α (ACACA isoform 1)98, 99. 30% (21 proteins) of the proteins meeting the ratio cut-off were proteasome subunits reflecting the ubiquitin affinity housed within the proteasome100. Ingenuity pathway analysis software revealed the entire proteasome subunit network being enriched upon proteasome inhibition (data not shown). Since this purification is non-denaturing, some enriched proteins upon MG132 may actually be indirectly interacting with ubiquitylated proteins; therefore, this method was not specifically enriching ubiquitylated proteins.  43  Figure 3.3 GST-S5a SILAC enrichment graph. Cells labeled with light amino acids were treated with DMSO and cells labeled with heavy amino acids were proteasome inhibited. Graph represents the enrichment ratio on a log2 scale versus the corresponding protein number. A subset of the proteins are highlighted. Proteins listed have a minimum of two peptides identified by Mascot. A trend-line for ratio versus protein number between 75 and 290 is depicted; deviation above the trend-line starts below protein number 73.  44  Table 3.2 GST-S5a SILAC ratios of significantly enriched proteins. MSQuant was used to calculate the enrichment ratio of heavy versus light peptide. Cells treated with MG132 and DMSO were labeled with heavy and light amino acids respectively. The cut-off ratio used for enrichment was 0.57.  Proteins from GST-S5a pull-down enriched Number of peptides upon proteasomal inhibition ANKRD5 Ankyrin repeat domain-containing protein 5
 MATR3 Matrin-3
 PSMB5 29 kDa protein
 LOC652826 similar to 26S proteasome ATPase subunit
 UBQLN2 Ubiquilin-2
 PSMC6 26S protease regulatory subunit S10B
 PSMD3 26S proteasome non-ATPase regulatory subunit 3
 COPA Coatomer subunit alpha
 MAGED1 Isoform 1 of Melanoma-associated antigen D1
 BAT3 Isoform 1 of Large proline-rich protein BAT3
 PSMB4 Proteasome subunit beta type-4
 PSMD2 26S proteasome non-ATPase regulatory subunit 2
 PSMA5 Proteasome subunit alpha type-5
 UBR4 Isoform 3 of E3 ubiquitin-protein ligase UBR4
 PSMB6 Proteasome subunit beta type-6
 SQSTM1 Isoform 1 of Sequestosome-1
 PSMD7 26S proteasome non-ATPase regulatory subunit 7
 PSMC5 26S protease regulatory subunit 8
 GNB2 Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2
 PSMD14 26S proteasome non-ATPase regulatory subunit 14
 DBN1 Isoform 1 of Drebrin
 PSMD1 Isoform 2 of 26S proteasome non-ATPase regulatory subunit 1
 PSMC2 26S protease regulatory subunit 7
 PSMD6 26S proteasome non-ATPase regulatory subunit 6
 RGNEF RGNEF protein
 SMC2 structural maintenance of chromosomes 2-like 1
 PSMB1 Proteasome subunit beta type-1
 ACACA Isoform 1 of Acetyl-CoA carboxylase 1
 PSMC1 26S protease regulatory subunit 4
 PSMD5 26S proteasome non-ATPase regulatory subunit 5
 PSMD13 proteasome 26S non-ATPase subunit 13 isoform 2
 PSMB3 Proteasome subunit beta type-3
 BIRC6 baculoviral IAP repeat-containing 6
 HUWE1 482 kDa protein
 ATXN2L Isoform 1 of Ataxin-2-like protein
 C12orf51 chromosome 12 open reading frame 51
 USP7 Ubiquitin carboxyl-terminal hydrolase 7
 HNRNPF Heterogeneous nuclear ribonucleoprotein F
 IPO5 importin 5
 PSMC3 26S protease regulatory subunit 6A
 PRKDC Isoform 2 of DNA-dependent protein kinase catalytic subunit
 HSPA1B;HSPA1A Heat shock 70 kDa protein 1
 PSMA7 Isoform 1 of Proteasome subunit alpha type-7
 UBR5 E3 ubiquitin-protein ligase UBR5
 C6orf174 Uncharacterized protein C6orf174
 AARS2 Probable alanyl-tRNA synthetase, mitochondrial
 NEFM neurofilament, medium polypeptide 150kDa isoform 1
 HNRNPH1 Heterogeneous nuclear ribonucleoprotein H
 - 69 kDa protein
 NEFH Neurofilament heavy polypeptide
 PCM1 Isoform 3 of Pericentriolar material 1 protein
 KIAA1618 Isoform 3 of Protein ALO17
 EIF4A1 Eukaryotic initiation factor 4A-I
 HERC2 Probable E3 ubiquitin-protein ligase HERC2
 HDAC2 histone deacetylase 2
 NPLOC4 Isoform 2 of Nuclear protein localization protein 4 homolog
 SMC3 Structural maintenance of chromosomes protein 3
 HYOU1 Hypoxia up-regulated protein 1
 KIF21A Isoform 1 of Kinesin-like protein KIF21A
 PARC Novel protein
 FAM115A Isoform 1 of Protein FAM115A
 EXOC4 Exocyst complex component 4
 VCP Transitional endoplasmic reticulum ATPase
 PCNA Proliferating cell nuclear antigen
 LOC442497;SLC3A2 solute carrier family 3
 UBC;UBB;RPS27A Ubiquitin C splice variant
 RPL23 60S ribosomal protein L23
 IKBKAP inhibitor of kappa light polypeptide gene enhancer in B-cells
 EIF3CL;EIF3C Eukaryotic translation initiation factor 3 subunit C
 ATP5B ATP synthase subunit beta, mitochondrial
 PPP2R1A Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A
 MCM3 DNA replication licensing factor MCM3
  1
 2
 4
 4
 3
 4
 3
 2
 2
 29
 3
 22
 1
 51
 2
 2
 1
 7
 1
 2
 3
 13
 7
 3
 3
 8
 3
 4
 6
 2
 2
 2
 1
 27
 1
 10
 12
 1
 1
 6
 14
 9
 1
 11
 1
 1
 12
 3
 2
 1
 10
 1
 5
 11
 1
 4
 9
 3
 2
 1
 5
 2
 30
 1
 2
 13
 1
 3
 7
 15
 2
 2
  Protein Mascot Log2(heavy/ score light) 40
 85
 220
 242
 121
 125
 150
 91
 165
 1698
 151
 1105
 79
 2539
 114
 85
 65
 298
 49
 76
 272
 734
 367
 140
 101
 332
 181
 182
 245
 73
 137
 100
 41
 1558
 60
 515
 545
 49
 59
 240
 759
 552
 40
 498
 51
 43
 690
 209
 93
 62
 480
 71
 288
 475
 35
 189
 446
 113
 82
 64
 195
 158
 1723
 60
 73
 873
 47
 111
 485
 740
 55
 148
  9.97
 3.13
 3.11
 2.94
 2.82
 2.51
 2.51
 2.50
 2.49
 2.47
 2.26
 2.17
 2.15
 2.15
 2.09
 2.06
 2.05
 2.04
 2.03
 2.01
 1.99
 1.99
 1.98
 1.88
 1.84
 1.83
 1.80
 1.76
 1.73
 1.70
 1.66
 1.62
 1.61
 1.61
 1.57
 1.54
 1.52
 1.51
 1.47
 1.46
 1.44
 1.38
 1.28
 1.28
 1.19
 1.18
 1.17
 1.17
 1.12
 1.04
 1.03
 0.98
 0.94
 0.91
 0.91
 0.90
 0.90
 0.86
 0.82
 0.80
 0.79
 0.77
 0.75
 0.73
 0.70
 0.69
 0.69
 0.65
 0.63
 0.62
 0.58
 0.57
  45  3.5 Direct immunoprecipitation of parkin binding partners We have carried out two ubiquitin purification procedures and analyzed the number of proteins by MS. The HBU approach revealed a bias towards highly abundant proteins along with a low number of total proteins. S5a affinity purification lead to the identification of many ubiquitin binding proteins, including the entire proteasome complex, suggesting a difficulty to unambiguously identify proteins that are ubiquitylated themselves. In the face of these non-ideal situations, we decided to employ direct parkin immunoprecipitation to capture interactors and identify them by MS, which had not been done before. Several parkin ligase substrates were first discovered by an initial positive co-immunoprecipitation and later confirmed by subsequent ubiquitylation assays35, 101103,  indicating that this approach may be suitable.  We used parkin N-terminally (amino-) tagged with the short amino acid FLAG sequence that is recognized by antibodies enabling single-step affinity purification65. We generated a stable cell line that genomically integrated the plasmid containing FLAGparkin and the geneticin resistance gene in order to obtain reliable and constant expression levels of the ectopic FLAG-parkin. By clonal dilution, we obtained ten pure populations of SH-SY5Y cells resistant to geneticin (G418). We were able to readily detect parkin in three clones (Figure 3.4A). Endogenous parkin was not detected due to very low levels of expression. We confirmed by immunofluorescence that expression of FLAG-parkin was homogenous in these three clonal populations (data not shown). We concluded that both the neomycin resistance cassette and the FLAG-parkin were integrated into euchromatin in these cells. Clone 4 was selected for subsequent  46  experiments due to the intermediate expression level of the ectopic protein. We reasoned that a large molar excess of the bait over its interacting partners (e.g. Clone 3) may lead to a higher rate of false negative identification by MS103. We performed FLAG-parkin immunoprecipitation using anti-FLAG sepharose, and captured about 50% of the ectopic protein (Figure 3.4B). We verified that the western blotting signal was specific to FLAG-parkin since anti-FLAG sepharose treated with SDS did not produce any signal (data not shown). Negligible amounts of FLAG-parkin were present in the lysis buffer washes (data not shown).  1  A  2  3  4  5  6  7  8  60 50 40  9  10 11  FLAG-parkin parkin  1  B 60 50 40 30  2  3  4 FLAG-parkin IgG light chain  20 Figure 3.4 Purification of FLAG-parkin. Cells cloned after integration of the FLAG-parkin plasmid were lysed and subjected to SDS-PAGE and immunoblotting with anti-parkin PRK8 antibody (A). Lane 1 untransfected, lanes 2-11 FLAG-parkin clones. The arrows indicate where FLAG-parkin and endogenous parkin would be expected to migrate. Clone 4 was chosen for subsequent experiments. Aliquots at different stages of the purification scheme were subjected to SDS-PAGE and immunoblotting with PRK8 antibody (B). Lane 1 (1x) input, lane 2 (0.33x) input, lane 3 (1x) unbound, lane 4 (5x) elution. Molecular weights in kDa are indicated to the right of each blot.  47  We used SILAC to differentiate between specific parkin interactors and non-specific background proteins (Figure 3.5). Two cell populations (2x107 cells each), one stably expressing FLAG-parkin and the other not, were labeled with light and heavy amino acids, respectively. Cells were lysed and processed separately by incubating each lysate with a batch of anti-FLAG sepharose to initially capture FLAG-parkin and then the beads were pooled during the first wash with lysis buffer. The rationale for not mixing the lysates during the immunoprecipitation is that dynamic interactors of parkin could display a high exchange rate resulting in a heavy labeled dynamic interactor exchanging with a light labeled dynamic interactor originally bound to FLAG-parkin derived from light labeled cells105. This rapid exchange would result to relative equal quantities of light and heavy labeled dynamic interactors bound to FLAG-parkin. In order to increase the number of proteins identified by MS, we first eluted the bound proteins into a weak interactor fraction by incubating the beads with detergent (1% Tween) and salt (500mM NaCl), and then eluted the remaining proteins into a strong interactor fraction by applying denaturing SDS detergent. The two protein mixtures were trypsin digested into peptides, then subjected to strong cation exchange chromatography into four fractions and analyzed by LC-MS/MS as previously described91. Assigned peak lists from each sample were pooled and ratios quantified by MSQuant. We identified 295 and 110 proteins from the weak interactor and strong interactor fractions respectively (Figure 3.6, Appendix Tables 3 and 4). FLAG-parkin was the most SILAC enriched protein and with a confident number of eight peptides identified. Parkin does not have a reported log2(heavy/light) ratio due to undetectable amounts of heavy peptides from the normal cell population relative to light peptides from FLAG-parkin 48  Figure 3.5 Quantitative mass spectrometry to determine parkin interactors. Schematic representation of the SILAC approach. SILAC was employed to differentiate between specific and background proteins. Specific interactors would have a heavy/light ratio < 1, non-specific having a ratio ~1.  over-expressing cells hence a heavy/light ratio of zero which cannot have a logarithmic value (Figure 3.7A). We used parkin derived from Rattus norvegicus cDNA which has unique peptides compared to human parkin peptides, hence each MS spectrum file was also searched against the rat database to identify and quantify these peptides. Based on a cutoff score of log2(heavy/light) = 1.00, we identified 24 potential parkin interactors (Appendix, Table 3). Candidate interactors included neuroendocrine chromograninsecretogranin family proteins that are synthesized in the ER and stored in secretory granules in the cell106 (Table 3.3; see also Figure 3.7B for representative spectrum of one peptide). The neuroendocrine candidate interactors were weakly interacting because they were absent in the second elution. DJ-1, a known parkin interactor, was identified in the final elution confirming the validity of the technique55. 49  Log2(heavy/light)  Figure 3.6 FLAG-parkin SILAC enrichment graph. Cells labeled with light amino acids were stably expressing FLAG-parkin and cells labeled with heavy amino acids were not expressing FLAG-parkin. Graph represents the enrichment ratio on a log2 scale of heavy/light SILAC labeling versus the corresponding protein number. A subset of the enriched proteins from the weak interactor fraction are highlighted.  50  A  Parkin  MSGECQSPDCPGTR  Mascot score = 38 MSQuant heavy/light ratio = N/A  secretogranin-2  B  ALEVIENLR  Mascot score = 64 MSQuant heavy/light ratio = 0.29  Figure 3.7 Relative intensity versus m/z of parkin and secretogranin-2 (A). Highlighted are peptide signatures belonging to parkin (A) and secretogranin-2 (B) from light amino acid labeled cells over-expressing FLAG-parkin. The peptide sequence for parkin is MSGECQSPDCPGTR with a monoisotopic mass of 791.31 Th. The peptide charge is +2 due to a spacing of 0.50 Th between isotopic peaks. The expected heavy labeled parkin peptide monoisotopic peak position is shown by the arrow at 794.31 Th as well as other indicated light/heavy monoisotopic peaks. Other spectra peaks are background signals that have the same retention time. The peptide sequence for secretogranin-2 is ALEVIENLR with a monoisotopic mass of 560.805 Th for the light peptide. The peptide charge is +2 due to a spacing of 0.50 Th between isotopic peaks.  51  Table 3.3 Enriched protein ratios from FLAG-parkin pull-down. n/a = not applicable. MSQuant manually corrected ratios are in fourth column. Proteins were either present in the weak interactor fraction or strong interactor fraction. Enriched proteins from FLAG-parkin DJ-1 secretogranin-3 chromogranin-A secretogranin-2 parkin  Protein Mascot Score 52 175 696 1688 379  Number of peptides 1 22 21 41 8  log2(heavy/ light) -0.83 -1.80 -1.36 -2.07 n/a  log2(heavy/light) manual correction -0.83 -2.12 -1.36 -2.07 n/a  Fraction strong weak weak weak strong  3.6 Capturing parkin interactors with cross-linking prior to immunoprecipitation We were concerned that parkin was interacting with unspecific proteins after lysis because the major interactors were granin family proteins, which typically reside in granules and has no domain in the cytoplasm. Moreover, only one peptide was identified for DJ-1, a known interactor, indicating that true interactors could rapidly dissociate. In order to reduce potential background binders, we exploited the approach developed by Vasilescu et al.107, in which formaldehyde was utilized to trap protein interactions in vivo. Formaldehyde is a small membrane permeable molecule, which can form cross-links between amine groups in close proximity such as between interacting proteins. The advantage of this technique is that transient interactors with a high koff rate can be preserved intact during the immunoprecipitation procedure enabling extensive washing to remove non-specific proteins bound to the target protein or resin107. Formaldehyde cross-links are reversible enabling proper identification of peptides by Mascot. 52  We established a cell line that expresses Myc-parkin since cross-linking masked the FLAG epitope. Higher molecular weight parkin signals were detected by SDS-PAGE and parkin immunoblotting from Myc-parkin expressing cells treated with formaldehyde suggesting entrapment of parkin in a covalent bond with binding partners (Figure 3.8A). This increase in complex preservation reached a maximum at 0.40% formaldehyde treatment so this concentration was used for subsequent experiments. Excessive crosslinking is not desirable because there would potentially be more non-specific proteins reacting with real parkin complexes. We next immunoprecipitated Myc-parkin complexes with anti-Myc 9E10 protein G magnetic beads. The pull-down efficiency of Myc-parkin was about 30% and was not affected by cross-linking (Figure 3.8B approximately equal parkin signals in the 3% eluate versus 1% input lane). The higher molecular weight parkin species disappeared along with an increase in uncross-linked parkin after incubating in high temperature reflecting the reversible nature of the formaldehyde cross-links and proteomics compatibility (Figure 3.8, lane 9). We purified Myc-parkin complexes from formaldehyde treated cells versus cells not expressing Myc-parkin (6x106 cells each condition), reversed the cross-links and subjected the protein mixture to LC-MS/MS. We identified only four parkin peptides, which was less than the typical FLAG immunoprecipitation. The low number of parkin peptides was not encouraging because it suggested many of the identified proteins may have cross-linked non-specifically or were contaminants still binding to the beads. We were also not able to identify any known parkin interactors; therefore, we decided not to pursue this approach.  53  A  B  1  2  3  4  5  6  7  8  9  cross-linked parkin complexes Myc-parkin parkin  Figure 3.8 Purification of Myc-parkin from cross-linked lysate. Optimization of formaldehyde concentration in cross-linking experiments (A). % formaldehyde is indicated above lanes. Marker positions in kDa are indicated on the right. Aliquots at different stages of the purification scheme were subjected to SDS-PAGE and immunoblotting with PRK8 antibody (B). Lane 1 (1x) untransfected input, lane 2 (1x) Myc-parkin cells input, lane 3 (1x) unbound, lane 4 wash, lane 5 wash, lane 6 (3x) 0.5% SDS elution, lane 7 (3x) 2% SDS elution, lane 8 (3x) 6% SDS elution, lane 9 (3x) 0.5% boiled elution.  54  3.7 Quantitative proteomic analysis of parkin interactors upon mitochondrial stress Parkin was shown to localize to the mitochondria after mitochondrial depolarization to promote mitophagy46, which is a significant leap in further establishing a connection between mitochondrial dysfunction and PD etiology. An important task will be deciphering the proteins mediating the parkin-mitophagy pathway and whether parkin’s ubiquitin ligase activity is required for downstream initiation of autophagy. We first assessed whether FLAG-parkin was able to act similarly to parkin, and indeed found that FLAG-parkin co-localized with the mitochondria upon 10µM CCCP treatment for 3 hours, but not DMSO treatment (Figure 3.9). A cross-section of the cytoplasm displayed overlap of the anti-parkin stain (green) and mitotracker stain (red) in cells treated with CCCP. We investigated parkin interactors upon mitochondrial membrane depolarization by performing co-immunoprecipitation with SILAC labeled cell lysate using the same procedure as described in Chapter 3.5. In this experiment, we compared light and heavy labeled cells treated with DMSO and CCCP, respectively (each expressing FLAG-parkin). We identified 1180 and 450 proteins in the weak and strong interactor fractions respectively (false positive rate ≤1%) that were quantified by MSQuant (Figure 3.10, Table 3.4, Appendix Tables 5 and 6). We identified 30 proteins enriched in CCCP treated cells including several actin related proteins (Table 3.6). Myosin regulatory light chain and myosin IX, a non-muscle unconventional myosin involved in signaling108 were both found enriched in CCCP treated cells and were readily identified with 13 and 32 peptides, respectively. Ion intensities of selected peptides for each protein in the full  55  scan (i.e. unfragmented) confirmed the proteins were enriched from heavy labeled cells (Figure 3.11). Mitochondrial apoptosis inducting factor (MAIF) was also enriched upon CCCP treatment suggesting specific stress on the mitochondria causing potential downstream signaling events. Only one peptide was identified for MAIF but the Mascot score was above 50, which was very high and strongly suggested the protein was present. The enriched proteins were all present in the final elution suggesting a strong binding interaction with FLAG-parkin. Parkin was identified with 23 peptides but with a de-enriched ratio from CCCP treated cells (not absent though). We confirmed the de-enrichment was based on loss of parkin solubility (Figure 3.12). This outcome may be due to parkin being recruited to autophagosomes at mitochondria upon mitochondrial membrane depolarization resulting in a higher sedimentation coefficient and pelleting during lysate clearance (Figure 3.9). Thus, several proteins interacting with parkin due to CCCP may have a similar de-enrichment. Cytochalasin D was used to validate the significance of the actin cytoskeleton in the parkin-mediated mitophagy pathway since the fungal metabolite inhibits actin polymerization in eukaryotic cells109. Immunofluorescence co-localization studies revealed that cytochalasin D prevented the full recruitment of parkin to mitochondria (Figure 3.13). In addition, several parkin punctuates were not observed to be colocalized with mitochondria and the parkin signal was much more diffuse.  56  Parkin  Mitotracker  Merge  Profile  DMSO  CCCP  Figure 3.9 Parkin recruitment to the mitochondria upon CCCP treatment. Mitotracker orange was used to stain mitochondria and PRK8 used to stain parkin. Profile graphs show relative signal intensities of parkin and mitochondria based on the selected profile depicted by the red line. Magnification is 63X and the blue scale bar represents 10µm.  57  Figure 3.10 FLAG-parkin CCCP SILAC enrichment graph. FLAG-parkin stable cells treated with DMSO or CCCP were labeled with light and heavy amino acids, respectively. Graph represents the enrichment ratio on a log2 scale of the heavy/light SILAC ratio versus the corresponding protein number. A subset of the enriched proteins from the strong interactor fraction are highlighted. Proteins listed have a minimum of two peptides identified by Mascot. A subset of proteins in the region underlined had ratios manually validated using MSQuant. MRLC2 and MYH9 actually have much greater ratios than 6, the graph maximum.  58  A  myosin IX ELESQISELQEDLESER Mascot score = 83 MSQuant heavy/light ratio = 5.35  B  myosin regulatory light chain GNFNYIEFTR Mascot score = 57 MSQuant heavy/light ratio = 6.50  Figure 3.11 Relative intensity versus m/z showing myosin-IX and myosin regulatory light chain peptides. Highlighted are peptide signatures belonging to myosin-IX (A) and myosin regulatory light chain (B) from light amino acid labeled cells treated with DMSO and heavy amino acid labeled cells treated with CCCP. The peptide sequence for myosin IX is ELESQISELQEDLESER with a monoisotopic mass of 1020.493 Th for the heavy peptide. The peptide charge is +2 due to a spacing of 0.50 Th between isotopic peaks. The peptide sequence for myosin regulatory light chain is GNFNYIEFTR with a monoisotopic mass of 634.316 Th for the heavy peptide. The peptide charge is +2 due to a spacing of 0.50 Th between isotopic peaks. Arrows indicate monoisotopic peaks for light/heavy peptides.  59  Table 3.4 Enriched proteins from CCCP treated FLAG-parkin lysate. MSQuant manually corrected ratios are in fourth column. Proteins were all present in the strong interactor fraction.  Proteins enriched with CCCP myosin regulatory light chain myosin IX MAIF myosin X drebrin parkin  Protein Number of log2(heavy log2(heavy/light) Mascot Score peptides /light) manual correct 700 13 3.54 19.14 1722 52 995 2024 1534  1 60 50 40  60 50 40  32 1 19 33 23  2  3  4  2.03 1.86 1.39 1.09 -2.86  5  6  7  16.95 1.88 1.57 1.09 -2.86  Fraction strong strong strong strong strong strong  8  FLAG-parkin  Ponceau S  Figure 3.12 Parkin solubility is reduced upon CCCP treatment. Cell were lysed as in Chapter 2.4.3 and debris pelleted at 4ºC, 16200g 10 min. Lane 1 to 4 represent cell lysates that have not be pelleted, lane 1 is 6 hours DMSO, lane 2 is 3 hours CCCP, lane 3 is 4.5 hours CCCP, lane 4 is 6 hours CCCP; lanes 5-8 represent cleared cell lysate after centrifugation, lane 5 is 6 hours DMSO, lane 6 is 3 hours CCCP, lane 7 is 4.5 hours CCCP and lane 8 is 6 hours CCCP. CCCP was used at 10µM. Membranes were incubated with Ponceau S total protein stain after transfer for 2 min, which shows relatively equal protein loading.  60  Parkin  Mitotracker  Merge  Profile  Parkin  Mitotracker  Merge  Profile  A  B  Figure 3.13 Cytochalasin D hampers parkin localization to mitochondria. FLAG-parkin cells were treated with either CCCP alone (A) or CCCP and cytochalasin D (B) and stained with PRK8 antibodies and mitotracker orange. A zoomed in box panel provides a detailed raw image of a profile graph. On the right, profile graphs show intensities of the parkin (green) and mitochondria (red) based on the selected profile in the zoomed panel of the merged images. There is no significant overlap between the red and green channels in cells with cytochalasin D treatment (B) relative to CCCP only (A). Magnification is 63X. Arrows represent stained parkin in which the corresponding mitochondrial signal is absent. Blue scale bar is 10µm.  61  CHAPTER 4 DISCUSSION  62  4.1 Ubiquitin purification methods for mammalian cells We tested different strategies to investigate parkin E3 ubiquitin ligase function, a protein implicated in juvenile Parkinsonism. We first sought to determine whether the enrichment of ubiquitylated proteins could allow us to identify proteins affected by parkin. Provided a good enrichment for low abundant poly-ubiquitylated proteins, ectopic parkin expression could reveal the presence of new ubiquitylated proteins that could correspond to its substrates due to increased parkin-mediated ubiquitylation and purification. The two methods used to purify the ubiquitin proteome were tandem purification of HBU conjugates from cells transfected with the HBU construct and purification of ubiquitin conjugates using S5a affinity chromatography. The coverage of a variety of low-abundance ubiquitylated proteins was essential when assessing putative substrates of parkin or any other E3 ligase because ligase substrates are often low in abundance such as ubiquitylated cell cycle proteins and parkin-associated endothelin-like receptor (Pael-R)35. Although the expression level of HBU was adequate relative to endogenous ubiquitin in a 2:3 ratio upon transfection, we found the incorporation of HBU into polyubiquitin chains was hindered leading to a low amount of purified ubiquitin conjugates. The molecular weight of the His8-biotinylation signal tag itself was relatively the same as ubiquitin; therefore, steric hinderance may have prevented ubiquitin conjugation enzyme machineries from properly recognizing and incorporating HBU. Furthermore, SILAC experiments with this approach110 in Dr. P. Kaiser’s laboratory (UC Irvine) yielded no specific enrichment of ubiquitin conjugates in cells with p97 impairment (which normally leads to an increase of ubiquitin conjugates; R. Deshaies, personal communication). 63  The low yield of ubiquitin conjugates was evident in the low number of peptides identified when subjecting the tandem affinity purified material for MS. There existed an unfavourable bias for enrichment of highly abundant ubiquitylated proteins such as ribosome subunits92 and histones111. α/β-tubulin were the only known parkin substrates identified, but these are also highly abundant proteins and would not serve as the ideal reference parkin substrates to be potentially enriched in a parkin over-expression experiment. Proteasome inhibition increased the number of identified proteins as expected, although 41 total proteins was not representative enough of the entire ubiquitin landscape. This result was drastically different than the ubiquitin pulldowns under denaturing conditions performed with yeast lysate, in which several hundred ubiquitylated proteins were identified including low-abundance cell cycle proteins like CDC569. We initially hoped the yield of ubiquitin conjugates from the transfected mammalian cells would be similar to the His6-biotin-ubiquitin expressing yeast described by Tagwerker et al.69, but due to unforeseen circumstances of impaired conjugation, this approach is not likely feasible. We also tested His8-ubiquitin but were not able to obtain a high purification yield due to lower expression levels. We are currently testing a construct harboring four copies of His8-ubiquitin in tandem (His8-ubiquitin-His8-ubiquitin-His8-ubiquitin-His8-ubiquitin) in mammalian cells. We hope this construct will offer higher expression and improved conjugation.  We were the first to employ GST-S5a affinity chromatography in the context of SILAC to determine ubiquitylated proteins specifically enriched upon proteasomal inhibition. The 64  GST-S5a chromatography experiment enabled purification of a greater number of proteins than the HBU approach but the data was confounded by a large quantity of ubiquitin binding proteins due to protein-protein interactions under non-denaturing conditions. Many proteasome subunits were enriched due to the ubiquitin binding domains of the proteasome regulatory cap, which may interact with ubiquitylated proteins bound to the resin under native conditions during the pull-down incubation. Proteins with ubiquitin-associated (UBA) domains such as ubiquilin-2112, sequestosome1113 and HUWE1114 were three of the top enriched proteins upon proteasomal inhibition reflecting the nature of secondary interactions with the S5a resin. We identified a total of 310 proteins, much more than the HBU approach; however, only 20% of the total were enriched upon proteasomal inhibition (with a low cutoff of 0.57) suggesting the actual proportion of ubiquitylated proteins in the pool was smaller. Detergents cannot be added to the pull-down procedure to reduce background since hydrophobic interactions between S5a UIM domains and ubiquitin would be eliminated under such conditions (T. Mayor; personal communication). There may also be enriched ubiquitylated proteins that are not proteasome substrates and these cannot be excluded. We identified α/βtubulin again as the only known parkin substrate. We estimated that it would be difficult to employ this method as a tool for sifting parkin substrates upon parkin over-expression based on a highly-abundant protein.  65  4.2 Searching for parkin interactors by direct immunoprecipitation We sought to identify parkin interactors by parkin immunoprecipitation and mass spectrometry after the HBU and S5a purifications gave a shallow coverage of the ubiquitin proteome. We employed direct FLAG-parkin immunoprecipitation as well as formaldehyde cross-linking to enrich for parkin interactors and determined that the former was more appropriate. Using this approach, we then assessed parkin’s function in the mitochondrial autophagosome pathway upon mitochondrial dysfunction by enriching for potential novel interactors. We successfully purified Myc-parkin formaldehyde cross-linked complexes. However, a high number of peptides from naturally abundant proteins such as actin and tubulin comprised most of the top identified proteins (data not shown). Identification of highly abundant proteins may be due to the nature of the formaldehyde cross-link as parkin is known to bind to actin and tubulin resulting in the pull-down of large cytoskeletal components under non-denaturing conditions. Other identified proteins in the immunoprecipitant that are naturally abundant such as heat shock proteins and neuronal cell specific proteins may be indicators of non-specific proteins either sticking to the beads or non-specific cross-linking into large complexes containing parkin. The overshadowing of parkin peptides by a significant number of other proteins will hamper the identification of potential novel interactors because such interactors should at most be as abundant as the bait parkin. There were only four parkin peptides identified which was a concern during candidate interactor screening of the Mascot data list because the bait was not present at a high enough level to provide enough confidence that other  66  proteins are true interactors. We were also not able to identify any known parkin interactors. We identified many more bait parkin peptides using the FLAG-parkin direct immunoprecipitation method enabling more confident screening of other enriched candidate interactors. We were able to detect DJ-1, a known parkin interactor in the strong interactor fraction, which supported the validity of the parkin pull-down method. Many enriched proteins in the immunoprecipited fractions however, were represented by only one peptide which is not a confident indicator of whether the protein is really present. Analysis of the weak interactor fraction revealed several interesting parkin interactor candidates from the neuroendocrine chromogranin-secretogranin family including secretogranin-2, secretogranin-3 and chromogranin A. These proteins are acid, soluble secretory proteins in vesicles of the neuroendocrine system106. They contain an N-terminal signal peptide for translocation into the ER lumen and possess both intracellular and extracellular functions. Intracellular functions include contribution to secretory granule formation upon immature vesicle budding from the trans-Golgi network and extracellular functions are due to granin-derived peptide functioning106. Peptides identified by Mascot do not include the transmembrane signal peptide region suggesting possible interaction with parkin after ER translocation. Parkin is not known to localize within the ER, thus the interaction may be induced non-physiologically after cell lysis. However, a clinical connection between chromogranin A and PD also occurs in which chromogranin A accumulates in Lewy bodies of the substantia nigra and is diminished from the cerebrospinal fluid from PD patients115. We are currently  67  investigating whether this result is physiologically relevant by co-localization and coimmunoprecipitation methods. We identified several actin related proteins from FLAG-parkin cells upon CCCP treatment and validated the significance of the actin cytoskeleton in the parkinmitophagy process with co-localization studies. The MS enriched hits included proteins from the myosin family and actin binding protein known as drebrin. Myosin proteins have a motor domain and bind actin in an ATP sensitive manner and generate force through ATP hydrolysis. The amino-terminal motor domain is linked to a carboxylterminal tail via a neck domain that serves as the binding site for myosin light chains enabling the neck region to swing like a lever arm108. Expression of at least a dozen “unconventional” non-muscle myosins have been documented suggesting a wide range of functions for actin-based motors in the cell116. Myosins may participate in signal transduction such as myosin IX, one of the highest enriched proteins from the final elution fraction with CCCP treatment. Myosin IX appears to be cytoplasmic and partly associated with membranes and the actin cytoskeleton (Figure 4.1). Myosin IX may participate in signal transduction as the tail domain has 30% sequence identity with GTPase activating proteins of the Rho subfamily of small G-proteins, which act like molecular switches being active in their GTP-bound conformation and inactive in their GDP-bound conformation108. The Rho family of GTP-binding proteins has been implicated in the regulation of actin-based motile processes116. Myosin IX may serve to inactivate Rho in order to enable actin remodeling to occur. The role of myosin IX in the recruitment of parkin to the mitochondria may be due to the redistribution of parkin from the actin filaments to the mitochondria upon myosin IX actin remodeling117. Myosin IX 68  may also influence the trafficking of damaged mitochondria along microtubules by regulating the mitochondrial outer-membrane resident Miro GTPase, a protein regulating mitochondria transport, to make the mitochondria more accessible by parkin118. Interestingly, PINK1 was recently found to form a complex with Miro suggesting it may also play a role in mitochondrial transport119. The actin cytoskeleton has also been reported to mediate selective autophagy in S. cerevisiae, confirming our observations120.  d  d  d  f  f  f  Figure 4.1 Myosin IX domains. The head domain interacts with actin and the GTPaseactivating protein domain GAP stimulates the GTPase activity of GTP-bound Rho. Figure was adapted from reference 108.  69  4.3 Concluding remarks We were able to specifically enrich for ubiquitylated proteins using tandem affinity purification of HBU conjugates and S5a affinity chromatography. The depth of coverage of the ubiquitin proteome based on these two approaches was still quite shallow resulting in an inadequacy to screen for parkin ubiquitin E3 ligase substrates. Future experiments to improve the enrichment of ubiquitin conjugates could combine the usage of a tetra-His8-ubiquitin with GST-S5a affinity chromatography. MRM may also be used to detect low abundance known parkin substrate peptides in the ubiquitin purified material as a control for observing enrichment upon parkin over-expression (Kast, J; personal communication). We were able to identify candidate parkin interactors from the chromograninsecretogranin neuroendocrine family of proteins using direct FLAG-parkin immunoprecipitation. We were also able to identify non-muscle unconventional signaling myosin participating in the process of parkin-mediated mitophagy. Future experiments would be to validate these MS hits by co-immunoprecipitation and/or co-localization immunofluorescence to show a direct parkin interaction.  70  CHAPTER 5 REFERENCES  71  1. Mayor, T., and Deshaies, R.J. (2005) Two-step affinity purification of multiubiquitylated proteins from Saccharomyces cerevisiae. Methods Enzymol., 399, 385-92. 2. Hershko, A. and Ciechanover, A. (1998) The ubiquitin system. Ann. Rev. Biochem., 425-79. 3. Pickart, C.M. (2004) Back to the future with ubiquitin. Cell, 116, 181-90. 4. Deng, L., et al. (2000) Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell, 103, 351-61. 5. Raiborg, C., Stenmark, H. (2009) The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins. Nature, 458, 445-52. 6. Henry, K.W. et al. (2003) Transcriptional activation via sequential histone H2B ubiquitylation and deubiquitylation, mediated by SAGA-associated Ubp8. Genes Dev., 17, 2648-63. 7. Kao, C.F., et al. (2004) Rad6 plays a role in transcriptional activation through ubiquitylation of histone H2B. Genes Dev., 18, 184-95. 8. Joo, H.Y., et al. (2007) Regulation of cell cycle progression and gene expression by H2A deubiquitination. Nature, 449, 1068-72. 9. Bergink, S., and Jentsch, S. (2009) Principles of ubiquitin and SUMO modifications in DNA repair. Nature., 458, 461-7. 10. Boutet, S.C., et al. (2007) Regulation of Pax3 by proteasomal degradation of monoubiquitinated protein in skeletal muscle progenitors. Cell, 130, 349-62. 11. Richly, H., et al. (2005) A series of ubiquitin binding factors connects CDC48/p97 to substrate multiubiquitylation and proteasomal targeting. Cell, 120, 73-84. 12. Wickner, S., et al. (1999) Posttranslational quality control: folding, refolding and degrading proteins. Science, 286, 1888-93. 13. Kruse, J.P., et al. (2009) Modes of p53 regulation. Cell, 137, 609-22. 14. Ross, C.A., and Poirier, M.A. (2004) Protein aggregation and neurodegenerative disease. Nat. Med., 10, S10-17. 15. Dawson, T.M., and Dawson, V.L., (2003) Molecular pathways of neurodegeneration in Parkinson’s disease. Science, 302, 819-22.  72  16. Ross, C.A., and Pickart, C.M. (2004) The ubiquitin-proteasome pathway in Parkinson’s disease and other neurodegenerative diseases. Trends Cell Biol., 14, 70311. 17. Thomas, B. and Beal, M.F. (2007) Parkinson’s disease. Hum. Mol. Genet., 16, R183-94. 18. Shults, C.W. (2006) Lewy bodies. Proc. Natl Acad. Sci. U.S.A., 103, 1661-8. 19. Kopito, R.R. (2000) Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol., 10, 524-30. 20. Vigouroux, S., Briand, M., and Briand, Y. (2004) Linkage between the proteasome pathway and neurodegenerative diseases and aging. Mol. Neurobiol., 30, 201-21. 21. Tanaka, Y. et al. (2001) Inducible expression of mutant alpha-synuclein decreases proteasome activity and increases sensitivity to mitochondria-dependent apoptosis. Hum. Mol. Genet., 10, 919-26. 22. Singleton, A.B. et al. (2003) alpha-synuclein locus triplication causes Parkinson’s disease. Science, 302, 841. 23. R. Lam, Y.A., et al. (2002) A proteasomal ATPase subunit recognizes the polyubiquitin degradation signal. Nature, 416, 763-7. 24. S. Weissman, A.M. (2001) Themes and variations on ubiquitylation. Nat. Rev. Mol. Cell Biol., 2, 169-78. 25. Kitada T., et al. (1998) Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature, 392, 605-8. 26. Lücking, C.B., et al. (2000) Association between early-onset Parkinson’s disease and mutations in the parkin gene. N. Engl. J. Med., 342, 1560-7. 27. Shimura, H., et al. (2000) Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase. Nature Genet., 25, 302-5. 28. Giasson, B.I., and Lee, V.M. (2001) Parkin and the molecular pathways of Parkinson’s disease. Neuron, 31, 885-8. 29. Hristova, V.A., et al. (2009) Identification of a novel Zn+2-binding domain in the autosomal recessive juvenile Parkinson-related E3 ligase parkin. J. Biol. Chem., 284, 14978-86. 30. Imai Y., et al. Parkin suppresses unfolded protein stress-induced cell death through its E3 ubiquitin-protein ligase activity. J. Biol. Chem., 275, 35661-4. 73  31. Petrucelli, L., et al. (2002) Parkin protects against the toxicity associated with mutant alpha-synuclein: proteasome dysfunction selectively affects catecholaminergic neurons. Neuron, 36, 1007-19. 32. Chung, K.K., et al. (2004) S-nitrosylation of parkin regulates ubiquitination and comprises parkin’s protective function. Science, 304, 1328-31. 33. Farrer, M., et al. (2001) Lewy bodies and parkinsonism in families with parkin mutations. Ann. Neurol., 293-300. 34. Olzmann, J.A., et al. (2007) Parkin-mediated K63-linked polyubiquitination targets misfolded DJ-1 to aggresomes via binding to HDAC6. J. Cell Biol., 178, 1025-38. 35. Imai, Y., et al. (2001) An unfolded putative transmembrane polypeptide, which can lead to endoplasmic reticulum stress, is a substrate of Parkin. Cell, 105, 891-902. 36. Fallon L., et al. (2006) A regulated interaction with the UIM protein Eps15 implicates parkin in EGF receptor trafficking and PI(3)K-Akt signalling. Nat. Cell. Biol., 8, 834-42. 37. Tsai, Y.C., et al. (2003) Parkin facilitates the elimination of expanded polyglutamine proteins and leads to preservation of proteasome function. J. Biol. Chem., 278, 2204455. 38. Cyr, D.M., et al. (2002) Protein quality control: U-box-containing E3 ubiquitin ligases join the fold. Trends Biochem. Sci., 27, 368-75. 39. Schapira, A.H. (2008) Mitochondria in the aetiology and pathogenesis of Parkinson’s disease. Lancet Neurol., 7, 97-109. 40. Jenner, P., (2003) Oxidative stress in Parkinson’s disease. Ann. Neurol., 53, S26. 41. Abou-Sleiman, P.M., Muqit, M.M., and Wood, N.W. (2006) Expanding insights of mitochondrial dysfunction in Parkinson’s disease. Nat. Rev. Neurosci., 7, 207-19. 42. Hardy, J., et al. (2006) Genetics of Parkinson’s disease and parkinsonism. Ann. Neurol., 60, 389-98. 43. Greene, J.C., et al. (2003) Mitochondrial pathology and apoptotic muscle degeneration in Drosophila parkin mutants. Proc. Natl. Acad. Sci. U.S.A., 100, 4078-83. 44. Whitworth, A.J., et al. (2005) Increased glutathione S-transferase activity rescues dopaminergic neuron loss in a Drosophila model of Parkinson’s disease. Proc. Natl. Acad. Sci. U.S.A., 102, 8024-29.  74  45. Pesah, Y., et al. (2004) Drosophila parkin mutants have decreased mass and cell size and increased sensitivity to oxygen radical stress. Development, 131, 2183-94. 46. Narendra, D. et al. (2008) Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J. Cell. Biol., 183, 795-803. 47. Valente, E.M., et al. (2004) Hereditary early-onset Parkinson’s disease is caused by mutations in PINK1. Science, 304, 1158-60. 48. Gautier, C.A., et al. (2008) Loss of PINK1 causes mitochondrial functional defects and increased sensitivity to oxidative stress. Proc. Natl. Acad. Sci. U.S.A., 105, 113649. 49. Clark, I.E., et al. (2006) Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin. Nature, 441, 1162-6. 50. Park, J., et al. (2006) Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin. Nature, 441, 1157-61. 51. Yang, Y., et al. (2006) Mitochondrial pathology and muscle and dopaminergic neuron degeneration caused by inactivation of Drosophila Pink1 is rescued by parkin. Proc. Natl. Acad. Sci. U.S.A., 103, 10793-8. 52. Yang, Y., et al. (2008) Pink1 regulates mitochondrial dynamics through interaction with the fission/fusion machinery. Proc. Natl. Acad. Sci. U.S.A., 105, 7070-5. 53. Darios, F., et al. (2003) Parkin prevents mitochondrial swelling and cytochrome c release in mitochondria-dependent cell death. Hum. Mol. Genet., 12, 517-26. 54. Zhou, C., et al. (2008) The kinase domain of mitochondrial PINK1 faces the cytoplasm. Proc. Natl. Acad. Sci. U.S.A., 105, 12022-7. 55. Xiong, H., et al. (2009) Parkin, PINK1, and DJ-1 form a ubiquitin E3 ligase complex promoting unfolded protein degradation. J. Clin. Invest., 119, 650-60. 56. Pridgeon, J.W., et al. (2007) PINK1 protects against oxidative stress by phosphorylating mitochondrial chaperone TRAP1. PLoS Biol., 5, 1494-03. 57. Plun-Favreau, H., et al. (2007) The mitochondrial protease HtrA2 is regulated by Parkinson’s disease-associated kinase PINK1. Nat. Cell Biol., 9, 1243-52. 58. McBride, H.M. (2008) Parkin mitochondria in the autophagosome. J. Cell Biol., 183, 757-9.  75  59. Deng, H., et al. (2008) The Parkinson’s disease genes pink1 and parkin promote mitochondrial fission and/or inhibit fusion in Drosophila. Proc. Natl. Acad. Sci. U.S.A., 105, 14503-8. 60. Karbowski, M., Neutzner, A., and Youle, R.J. (2007) The mitochondrial E3 ubiquitin ligase MARCH5 is required for Drp1 dependent mitochondrial division. J. Cell Biol., 178, 71-84. 61. Li, W., et al. (2008) Genome-wide and functional annotation of human E3 ubiquitin ligases identifies MULAN, a mitochondrial E3 that regulates the organell’es dynamics and signaling. PLoS ONE, 3, e1487. 62. Yonashiro, R., et al. (2006) A novel mitochondrial ubiquitin ligase plays a critical role in mitochondrial dynamics. EMBO J., 25, 3618-26. 63. Tomlinson, E., et al. (2007) Methods for the purification of ubiquitinated proteins. Proteomics, 7, 1016-22. 64. Kirkpatrick, D.S., Gerber, S.A., and Gygi, S.P. (2005) Weighing in on ubiquitin: the expanding role of mass-spectrometry-based proteomics. Nat. Cell Biol., 7, 750-7. 65. Terpe, K. (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl. Microbiol. Biotechnol., 60, 523-33. 66. Beers, E.P., and Callis, J. (1993) Utility of polyhistidine-tagged ubiquitin in the purification of ubiquitin-protein conjugates and as an affinity ligand for the purification of ubiquitin-specific hydrolases. J. Biol. Chem., 268, 1645-9. 67. Callis, J., and Ling, R. (2005) Preparation, characterization, and use of tagged ubiquitins. Methods Enzymol., 399, 51-64. 68. Peng, J., et al. (2003) A proteomics approach to understanding protein ubiquitination. Nat. Biotechnol., 21, 921-6. 69. Tagwerker, C., et al. (2006) A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in-vivo cross-linking. Mol. Cell. Proteomics., 5, 737-48. 70. Mayor, T., et al. (2007) Quantitative profiling of ubiquitylated proteins reveals proteasome substrates and the substrate repertoire influenced by the Rpn10 receptor pathway. Mol. Cell. Proteomics., 6, 1885-95. 71. Mayor, T., et al. (2007) Analysis of polyubiquitin conjugates reveals that the Rpn10 substrate receptor contributes to the turnover of multiple proteasome targets. Mol. Cell. Proteomics., 4, 741-51.  76  72. Gururaja, T., et al. (2003) Multiple functional categories of proteins identified in an in vitro cellular ubiquitin affinity extract using shotgun peptide sequencing. J. Proteome Res., 2, 394-04. 73. Matsumoto, M., et al. (2005) Large-scale analysis of the human ubiquitin-related proteome. Proteomics, 5, 4145-51. 74. Bennett, E.J., et al. (2007) Global changes to the ubiquitin system in Huntington’s disease. Nature, 448, 704-8. 75. Vasilescu, J., et al. (2005) Proteomic analysis of ubiquitinated proteins from human MCF-7 breast cancer cells by immunoaffinity purification and mass spectrometry. J. Proteome Res., 4, 2192-200. 76. Kirkpatrick, D.S., et al. (2005) Proteomic identification of ubiquitinated proteins from human cells expressing His-tagged ubiquitin. Proteomics, 5, 2104-11. 77. Deveraux, Q., et al. (1994) A 26 S protease subunit that binds ubiquitin conjugates. J. Biol. Chem., 269, 7059-61. 78. Young, P. et al. (1998) Characterization of two polyubiquitin binding sites in the 26S protease subunit 5a. J. Biol. Chem., 273, 5461-7. 79. Layfield, R. et al. (2001) Purification of poly-ubiquitinated proteins by S5a-affinity chromatography. Proteomics, 1, 773-7. 80. Weekes, J., et al. (2003) Hyperubiquitination of proteins in dilated cardiomyopathy. Proteomics. 3, 208-16. 81. Kingston, R.E., Chen, C.A., and Okayama, H. (2003) Calcium phosphate transfection. Curr. Protoc. Cell Biol., 20, 20.3. 82. Wessel, D. and Flügge, U.I. (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem., 138, 141-3. 83. Ishihama, Y., Rappsilber, J., and Mann, M. (2006) Modular Stop and Go Extraction Tips with Stacked Disks for Parallel and Multidimensional Peptide Fractionation in Proteomics. J. Proteome Res., 5, 988-94. 84. Rappsilber, J., and Mann, M. (2003) Stop and Go Extraction Tips for Matrix-Assisted Laser Desorption/Ionization, Nanoelectrospray, and LC/MS Sample Pretreatment in Proteomics. Anal. Chem., 75, 663-70. 85. Rappsilber, J., Mann, M., and Ishihama, Y. (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips, Nat. Protocols, 2, 1896-906. 77  86. Shevchenko, A., et al. (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc., 1, 2856-60. 87. Winkler, C., et al. (2007) Silver- and coomassie-staining protocols: detection limits and compatibility with ESI MS. Electrophoresis, 28, 2095-9. 88. Olsen, J.V., et al. (2005) Parts per Million Mass Accuracy on an Orbitrap Mass Spectrometer via Lock Mass Injection into a C-trap. Mol. Cell. Proteomics, 4, 2010–21. 89. West, A.B., et al. (2003) Parkin is not regulated by the unfolded protein response in human neuroblastoma cells. Neurosci. Lett., 341, 139-42. 90. de Boer, E., et al. (2003) Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice. Proc. Natl. Acad. Sci. U.S.A., 100, 7480-5. 91. Chan, Q.W.T., Howes, C.G., and Foster, L.J. (2006) Quantitative comparison of caste differences in honeybee hemolymph. Mol. Cell. Proteomics, 5, 2252-62. 92. Kraft, C., et al. (2008) Mature ribosomes are selectively degraded upon starvation by an autophagy pathway requiring the Ubp3p/Bre5p ubiquitin protease. Nat. Cell Biol., 10, 602-10. 93. Ren, Y., Zhao, J., Feng, J. (2003) Parkin binds to alpha/beta tubulin and increases their ubiquitination and degradation. J. Neurosci., 23, 3316-24. 94. Mann, M. (2006) Functional and quantitative proteomics using SILAC. Nat. Rev. Mol. Cell. Biol., 7, 952-8. 95. Cheng, I.H., Roberts, L.A., and Tye, B.K., et al. (2002) Mcm3 is polyubiquitinated during mitosis before establishment of the pre-replication complex. J. Biol. Chem., 277, 41706-14. 96. Hoege, C., et al. (2002) Rad6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature, 419, 135-41. 97. Yamamoto, T., et al. (2004) Degradation of proliferating cell nuclear antigen by 26S proteasome in rice (Oryza sativa L.). Planta, 218, 640-6. 98. Qi, L., et al. (2006) TRB3 links the E3 ubiquitin ligase COP1 to lipid metabolism. Science, 312, 1763-6.  78  99. Ma, J., et al. (2008) Aldo-keto reductase family 1 B10 affects fatty acid synthesis by regulating the stability of acetyl-CoA carboxylase-alpha in breast cancer cells. J. Biol. Chem., 283, 3418-23. 100. Husnjak, K., et al. (2008) Proteasome subunit Rpn13 is a novel ubiquitin receptor. Nature, 453, 481-8. 101. Chung, K.K., et al. (2001) Parkin ubiquitinates the alpha-synuclein-interacting protein, synphilin-1: implications for Lewy-body formation in Parkinson disease. Nat. Med., 7, 1144-50. 102. Huynh, D.P., et al. (2003) The autosomal recessive juvenilie Parkinson disease gene product, parkin, interacts with and ubiquitinates synaptotagmin XI. Hum. Mol. Genet., 12, 2587-97. 103. Dehvari, N., et al. (2008) Parkin-mediated ubiquitination regulates phospholipase C-gamma1. J. Cell. Mol. Med., “Postprint”. 104. Gingras, A.C. (2007) Analysis of protein complexes using mass spectrometry. Nat. Rev. Mol. Cell. Biol., 8, 645-54. 105. Wang, X., and Huang, L. (2008) Identifying dynamic interactors of protein complexes by quantitative mass spectrometry. Mol. Cell. Proteomics., 7, 46-57. 106. Taupenot, L., Harper, K.L., and O’Connor, D.T. (2003) The ChromograninSecretogranin Family. N. Engl. J. Med., 348, 1134-49. 107. Vasilescu, J., Guo, X., and Kast, J. (2004) Identification of protein-protein interactions using in-vivo cross-linking and mass spectrometry. Proteomics., 4, 3845-54. 108. Bähler, M. (2000) Are class III and class IX myosins motorized signaling molecules? Biochim. Biophys. Acta., 1496, 52-9. 109. Casella, J.F., Flanagan, M.D., and Lin, S. (1981) Cytochalasin D inhibits actin polymerization and induces depolymerization of actin filaments formed during platelet shape change. Nature, 293, 302-5. 110. Meierhofer, D. et al. (2008) Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry. J. Proteome Res., 7, 4566-76. 111. Wang, H., et al. (2004) Role of histone H2A ubiquitination in Polycomb silencing. Nature, 431, 873-8. 112. Regan-Klapisz, E., et al. (2005) Ubiquilin recruits Eps15 into ubiquitin-rich aggregates via a UIM-UBL interaction. J. Cell Sci., 118, 4437-50.  79  113. Evans, C.L., et al. (2008) Conformation and dynamics of the three-helix bundle UBA domain of p62 from experiment and simulation. Proteins, 71, 227-40. 114. Zhao, X., et al. (2008) The HECT-domain ubiquitin ligase Huwe1 controls neural differentiation and proliferation by destabilizing the N-Myc oncoprotein. Nat. Cell Biol., 10, 643-53. 115. Munoz, D.G. (1991) Chromogranin A-like immunoreactive neurites are major constituents of senile plaques. Lab. Invest., 64, 826-32. 116. Mermall, V., Post, P.L., and Moosker, M.S. (1998) Unconventional myosins in cell movement, membrane traffic, and signal transduction. Science, 279, 527-33. 117. Huynh, D.P. (2000) Parkin is associated with actin filaments in neuronal and nonneural cells. Ann. Neuro., 48, 737-44. 118. Reis, K., Fransson, Å., and Aspenström, P. (2009) The Miro GTPases: At the heart of the mitochondrial transport machinery. FEBS Lett., 583, 1391-8. 119. Weihofen, A., et al. (2009) Pink1 forms a multiprotein complex with Miro and Milton, linking Pink1 function to mitochondrial trafficking. Biochemistry, 48, 2045-52. 120. Reggiori, F., et al. (2005) The actin cytoskeleton is required for selective types of autophagy, but no nonspecific autophagy, in the yeast Saccharomyces cerevisiae. Mol. Biol. Cell., 16, 5843-56. 121. Zhang, Y., et al. (2000) Parkin functions as an E2-dependent ubiquitin-protein ligase and promotes the degradation of the synaptic vesicle-associated protein, CDCrel1. Proc. Natl. Acad. Sci. U.S.A., 97, 13354-9. 122. Shimura, H., et al. (2001) Ubiquitination of a new form of alpha-synuclein by parkin from human brain: implications for Parkinson’s disease. Science, 293, 263-9. 123. Ko, H.S., et al. (2005) Accumulation of the authentic parkin substrate aminoacyltRNA synthetase cofactor, p38/JTV-1. J. Neurosci., 31, 7968-78. 124. Staropoli, J.F., et al. (2003) Parkin is a component of an SCF-like ubiquitin ligase complex and protects postmitotic neurons from kainite excitotoxicity. Neuron, 37, 73549. 125. Uchiki, T., et al. (2009) The ubiquitin-interacting motif protein, S5a, is ubiquitinated by all types of ubiquitin ligases by a mechanism different from typical substrate recognition. J. Biol. Chem., 284, 12622-32.  80  126. Fukae, J., et al. (2009) Programmed cell death-2 isoform 1 is ubiquitinated by parkin and increased in the substantia nigra of patients with autosomal recessive Parkinson’s disease. FEBS Lett., 583, 521-5. 127. Joch, M., et al. (2007) Parkin-mediated monoubiquitination of the PDZ protein PICK1 regulates the activity of acid-sensing ion channels. Mol. Biol. Cell., 18, 3105-18. 128. Ko, H.S., et al. (2006) Identification of far upstream element-binding protein-1 as an authentic Parkin substrate. J. Biol. Chem., 281, 16193-6. 129. Lim, M.K., et al. (2007) Parkin interacts with LIM Kinase 1 and reduces its cofilinphosphorylation activity via ubiquitination. Exp. Cell. Res., 313, 2858-74. 130. Huynh, D.P., et al. (2007) Parkin is an E3 ubiquitin-ligase for normal and mutant ataxin-2 and prevents ataxin-2-induced cell death. Exp. Neurol., 203, 531-41. 131. Um, J.W., et al. (2006) Parkin ubiquitinates and promotes the degradation of RanBP2. J. Biol. Chem., 281, 3595-603. 132. Dächsel, J.C., et al. (2005) Parkin interacts with the proteasome subunit alpha4. FEBS Lett., 579, 3913-9. 133. Zhong, L., et al. (2005) RING finger ubiquitin-protein isopeptide ligase Nrdp1/FLRF regulates parkin stability and activity. J. Biol. Chem., 280, 9425-30. 134. Choi, P., et al. (2003) SEPT_v2 is a parkin-binding protein. Mol. Brain Res., 117, 179-89. 135. Staropoli, J.F., et al. (2003) Parkin is a component of an SCF-like ubiquitin ligase complex and protects postmitotic neurons from kainate excitotoxicity. Neuron, 37, 73549. 136. Imai, Y., et al. (2002) CHIP is associated with Parkin, a gene responsible for familial Parkinson’s disease, and enhances its ubiquitin ligase activity. Mol. Cell., 10, 55-67. 137. Fallon, L., et al. (2002) Parkin and CASK/LIN-2 associate via a PDZ-mediated interaction and are co-localized in lipid rafts and postsynaptic densities in brain. J. Biol. Chem., 277, 486-91. 138. Shiba, K., et al. (2009) Parkin stabilizes PINK1 through direct interaction. Biochem. Biophys. Res. Commun., 383, 331-5. 139. Um, J.W., and Chung, K.C. (2006) Functional modulation of parkin through physical interaction with SUMO-1. J. Neurosci. Res., 84, 1543-54.  81  140. Yang, F., et al. (2005) Parkin stabilizes microtubules through strong binding mediated by three independent domains. J. Biol. Chem., 280, 17154-62. 141. Jiang, Q., Ren, Y., and Feng, J. (2008) Direct binding with histone deacetylase 6 mediates the reversible recruitment of parkin to the centrosome. J. Neurosci., 28, 12993-02. 142. Matsuda, N., et al. (2006) Diverse effects of pathogenic mutations of Parkin that catalyze multiple monoubiquitylation in vitro. J. Biol. Chem., 281, 3204-9. 143. Rubinsztein, D.C. (2006) The roles of intracellular protein-degradation pathways in neurodegeneration. Nature, 443, 780-6.  82  APPENDIX  83  Table 1. Known parkin substrates (A) and interactors that are not ligase targets (B). References are provided for each protein.  A  Known parkin substrate CDCrel-1 synphilin-1 glycosylated alpha-synuclein Pael-R alpha and beta tubulin synaptotagmin XI aminoacyl-tRNA synthetase cofactor cyclin E S5a programmed cell death-2 isoform 1 phospholipase C-gamma1 PICK1 far upstream element-binding protein-1 LIM kinase 1 Eps15 Ataxin-2 RanBP2  Reference 121 101 122 12 93 102 123 124 125 125 103 127 128 129 36 130 131  B  Known parkin non-substrate interactor proteasome subunit alpha4 Nrdp1/FLRF SEPT5_v2 SCF-like ubiquitin ligase complex CHIP CASK/LIN-2 PINK1 SUMO-1 microtubules DJ-1 HDAC6  Reference 132 133 134 135 136 137 138 139 140 55 141  84  A  647.4006 729.4095 918.4559 919.4399 962.5225 966.4957 1031.5797 1032.5637 1038.5094 1043.6352 1047.5746 1066.6135 1090.5692 1093.563 1109.5579 1176.6503 1177.5761 1177.6343 1178.5601 1181.6768 1193.5710 1193.5710 1194.5550 1195.5656 1211.5605 1236.5656 1350.737 1418.7843 1429.818 1434.7792 1440.75 1459.7783 1460.7623 1477.7195 1493.7144 1515.7966 1520.7140 1522.7740 1527.778 1528.7621 1531.7916 1536.7090 1565.7685 1602.7646 1603.7487 1618.7596 1709.8519 1710.8359 1752.8465 1753.8305 1764.9006 1786.9200 1800.9403 1828.9306 1947.9425 1948.9265 1949.9106 1972.0517 2129.1480 2130.1321 2205.0801 2207.0481 2248.0746 2268.1313 2288.1999 2319.1077 2325.1331 2329.2264 2356.1991 2357.1831 2399.1936 2401.2588 2486.3479 2827.4828 3013.4662 3015.4343 3107.5663 3270.615 3654.9178  LIFAGK LPEMLK NGDFLPTR NGDFLPTR SPILGYWK ITFCTGIR LTQSMAIIR LTQSMAIIR EGIPPDQQR VAHLALKHR LTQSMAIIR ESTLHLVLR LTQSMAIIR MSPILGYWK MSPILGYWK LTAFVNTLNGK NAMGSLASQATK LTAFVNTLNGK NAMGSLASQATK RIEAIPQIDK NAMGSLASQATK NAMGSLASQATK NAMGSLASQATK MTISQQEFGR MTISQQEFGR NAMGSLASQATK MSPILGYWKIK VDFLSKLPEMLK IEAIPQIDKYLK VDFLSKLPEMLK DFETLKVDFLSK LIFAGKQLEDGR LIFAGKQLEDGR NAMGSLASQATKDGK NAMGSLASQATKDGK AEISMLEGAVLDIR NAMGSLASQATKDGK IQDKEGIPPDQQR VGSPVEDNEKDLVK VGSPVEDNEKDLVK AEISMLEGAVLDIR NAMGSLASQATKDGK IQDKEGIPPDQQR YIADKHNMLGGCPK YIADKHNMLGGCPK YIADKHNMLGGCPK LQAQQDAVNIVCHSK LQAQQDAVNIVCHSK LQAQQDAVNIVCHSK LQAQQDAVNIVCHSK AKIQDKEGIPPDQQR TITLEVEPSDTIENVK ERAEISMLEGAVLDIR TITLEVEPSDTIENVK VNVDIINFGEEEVNTEK VNVDIINFGEEEVNTEK VNVDIINFGEEEVNTEK IIAFVGSPVEDNEKDLVK TLSDYNIQKESTLHLVLR TLSDYNIQKESTLHLVLR EKVNVDIINFGEEEVNTEK EKVNVDIINFGEEEVNTEK EKVNVDIINFGEEEVNTEK LLLEYLEEKYEEHLYER TLTGKTITLEVEPSDTIENVK AAAASAAEAGIATTGTEDSDDALLK YIAWPLQGWQATFGGGDHPPK TLTGKTITLEVEPSDTIENVK KFELGLEFPNLPYYIDGDVK KFELGLEFPNLPYYIDGDVK KFELGLEFPNLPYYIDGDVK TLTGKTITLEVEPSDTIENVK TLTGKTITLEVEPSDTIENVKAK QLEDGRTLSDYNIQKESTLHLVLR SNPENNVGLITLANDCEVLTTLTPDTGR SNPENNVGLITLANDCEVLTTLTPDTGR VNVDIINFGEEEVNTEKLTAFVNTLNGK TRSNPENNVGLITLANDCEVLTTLTPDTGR HLTMQIFVKTLTGKTITLEVEPSDTIENVK  B  776.3817 1104.6655 1242.6608 1302.6092 1591.7114 1620.709 1980.8775 2241.9947  FSGVPDR LLIYKVSNR VNSAAFPAPIEK FSGSGSGTDFTLK QNGVLNSWTDQDSK SLTSEDSAVYYCAR NTQPIMNTNGSYFVYSK DSTYSMSSTLTLTKDEYER  Table 2. Exclusion list for anti-FLAG immunoglobulin chain peptides (A) and GSTS5a peptides (B). The left column is the m/z of the corresponding excluded peptide. Different variable modifications assigned to peptides may result in more than one m/z per peptide sequence such as a regular versus Met oxidized peptide.  85  Table 3. FLAG-parkin pull-down proteins with SILAC ratios – weak interactor fraction Proteins eluted with mild detergent and salt (weak parkin interactors) NPY Neuropeptide Y
 HMGB3 High mobility group protein B3
 C9orf86 Isoform 2 of Putative GTP-binding protein Parf
 TPM1 Tropomyosin isoform
 SCG2 Secretogranin-2
 HNRNPUL1 Isoform 1 of Heterogeneous nuclear ribonucleoprotein U-like protein 1
 SCG3 Secretogranin-3
 C1orf144 Isoform 4 of UPF0485 protein C1orf144
 NPDC1 Neural proliferation differentiation and control protein 1
 BAG4 BAG family molecular chaperone regulator 4
 FAM3C Protein FAM3C
 EIF4EBP1 Eukaryotic translation initiation factor 4E-binding protein 1
 CHGA Chromogranin-A
 HN1L Isoform 1 of Hematological and neurological expressed 1-like protein
 PPP1R14B Protein phosphatase 1 regulatory subunit 14B
 BASP1 Brain acid soluble protein 1
 PTTG1IP Uncharacterized protein PTTG1IP
 COX7A2 Uncharacterized protein COX7A2
 C19orf43 Uncharacterized protein C19orf43
 RPL12 Isoform 2 of 60S ribosomal protein L12
 VGF VGF nerve growth factor inducible precursor
 LOC389842 similar to RanBP1
 SSNA1 Sjoegren syndrome nuclear autoantigen 1
 PIN4 protein (peptidyl-prolyl cis/trans isomerase) NIMA-interacting, 4
 TMSB10 Thymosin beta-10
 TTYH3 Isoform 1 of Protein tweety homolog 3
 MRPS36 28S ribosomal protein S36, mitochondrial
 C14orf166 UPF0568 protein C14orf166
 HNRNPU Isoform Long of Heterogeneous nuclear ribonucleoprotein U
 C17orf49 20 kDa protein
 MYCBP C-Myc-binding protein
 LOC348262 hypothetical protein LOC348262
 CRIP2 Cysteine-rich protein 2
 FAM107B cDNA FLJ45505 fis, clone BRTHA2020642, weakly similar to DRR1 protein
 LSM4 U6 snRNA-associated Sm-like protein LSm4
 EIF4H Similar to mKIAA0038 protein
 TAGLN2 Transgelin-2
 RPL36 60S ribosomal protein L36
 ANP32E Acidic leucine-rich nuclear phosphoprotein 32 family member E
 KIAA0515 hypothetical protein LOC84726
 NDUFA5 Putative uncharacterized protein DKFZp781K1356
 GTF2A1 Isoform 42 kDa of Transcription initiation factor IIA subunit 1
 XTP3TPA XTP3-transactivated gene A protein
 HDGF Hepatoma-derived growth factor
 RPLP2 60S acidic ribosomal protein P2
 HN1 Isoform 1 of Hematological and neurological expressed 1 protein
 PFDN4 Prefoldin subunit 4
 PABPC1 Isoform 1 of Polyadenylate-binding protein 1
 - cDNA FLJ44434 fis, clone UTERU2019491, moderately similar to Homo sapiens 41-kDa phosphoribosylpyrophosphate synthetase-associated protein
 CARHSP1 Calcium-regulated heat stable protein 1
 PGRMC2 Membrane-associated progesterone receptor component 2
 FAM162A UPF0389 protein FAM162A
 BCAP31 B-cell receptor-associated protein 31
 MAPT Isoform Tau-A of Microtubule-associated protein tau
 RPL38 8 kDa protein
 SCAMP3 secretory carrier membrane protein 3 isoform 1
 RPL8 60S ribosomal protein L8
 GADD45GIP1 Growth arrest and DNA-damage-inducible proteins-interacting protein 1
 PSME3 Isoform 1 of Proteasome activator complex subunit 3
 PDAP1 28 kDa heat- and acid-stable phosphoprotein
 ATP5J ATP synthase-coupling factor 6, mitochondrial
 SF3B2 splicing factor 3B subunit 2
 ST13 Hsc70-interacting protein
 MRPL12 39S ribosomal protein L12, mitochondrial
 RPS25 40S ribosomal protein S25
 FAU 40S ribosomal protein S30
 EIF3J Eukaryotic translation initiation factor 3 subunit J
 LYRM7 LYR motif-containing protein 7
 VAMP3 Vesicle-associated membrane protein 3
 CPLX2 Complexin-2
 UBE2L3 Ubiquitin-conjugating enzyme E2 L3
 RPS10 40S ribosomal protein S10
 PEBP1 Phosphatidylethanolamine-binding protein 1
 PRKCSH Glucosidase 2 subunit beta
 TPM4 Isoform 1 of Tropomyosin alpha-4 chain
 RPE Isoform 1 of Ribulose-phosphate 3-epimerase
 EDF1 Isoform 1 of Endothelial differentiation-related factor 1
 - 78 kDa protein
 SFRS3 Splicing factor, arginine/serine-rich 3
 SFRS7 Isoform 1 of Splicing factor, arginine/serine-rich 7
 SRP14 Signal recognition particle 14 kDa protein
  Protein Mascot Score  # of peptides  log2(heavy /light)  52
 31
 1688
 90
 48
 108
 175
 74
 71
 50
 163
 696
 343
 201
 47
 240
 93
 72
 121
 48
 874
 26
 104
 99
 253
 61
 179
 127
 99
 78
 49
 132
 518
 99
 97
 48
 356
 74
 52
 48
 210
 160
 434
 420
 1721
 448
 404
 289
  1
 1
 2
 2
 41
 3
 5
 2
 1
 1
 2
 8
 21
 6
 1
 5
 3
 1
 3
 1
 22
 1
 2
 2
 6
 1
 5
 2
 2
 1
 1
 4
 6
 2
 2
 1
 9
 1
 1
 1
 4
 3
 10
 8
 32
 9
 10
 6
  -3.20
 -2.71
 -2.17
 -2.09
 -2.07
 -1.89
 -1.80
 -1.79
 -1.61
 -1.61
 -1.54
 -1.38
 -1.36
 -1.35
 -1.30
 -1.22
 -1.22
 -1.20
 -1.19
 -1.17
 -1.16
 -1.16
 -1.08
 -1.07
 -1.03
 -1.02
 -1.00
 -1.00
 -1.00
 -0.98
 -0.97
 -0.91
 -0.90
 -0.90
 -0.88
 -0.85
 -0.83
 -0.83
 -0.82
 -0.81
 -0.80
 -0.75
 -0.74
 -0.74
 -0.74
 -0.73
 -0.70
 -0.69
  434
 216
 66
 79
 99
 675
 47
 201
 69
 51
 112
 624
 145
 87
 670
 202
 465
 287
 343
 76
 218
 57
 254
 451
 1423
 69
 961
 180
 129
 92
 661
 82
 288
  7
 4
 1
 1
 2
 17
 1
 5
 2
 1
 2
 15
 5
 2
 17
 6
 12
 10
 9
 2
 4
 1
 6
 10
 29
 2
 24
 2
 3
 2
 18
 3
 4
  -0.69
 -0.68
 -0.68
 -0.67
 -0.66
 -0.65
 -0.65
 -0.63
 -0.60
 -0.60
 -0.59
 -0.59
 -0.59
 -0.57
 -0.57
 -0.56
 -0.55
 -0.55
 -0.54
 -0.53
 -0.53
 -0.53
 -0.52
 -0.51
 -0.51
 -0.50
 -0.50
 -0.50
 -0.50
 -0.49
 -0.48
 -0.48
 -0.47
  86  Proteins eluted with mild detergent and salt (weak parkin interactors) PFDN6 Prefoldin subunit 6
 NUCB1 Nucleobindin-1
 UBAP2L Isoform 2 of Ubiquitin-associated protein 2-like
 HMGB1 High mobility group protein B1
 RPS19 40S ribosomal protein S19
 RPL23A;hCG_16001 60S ribosomal protein L23a
 CHCHD8 Isoform 2 of Coiled-coil-helix-coiled-coil-helix domain-containing protein 8
 LRRC59 Leucine-rich repeat-containing protein 59
 STMN1 Stathmin
 RPS13 40S ribosomal protein S13
 PNPO 19 kDa protein
 RANBP1 Ran-specific GTPase-activating protein
 MMAB Cob(I)yrinic acid a,c-diamide adenosyltransferase, mitochondrial
 NOL3 Isoform 1 of Nucleolar protein 3
 RPLP1 60S acidic ribosomal protein P1
 TPM3 tropomyosin 3 isoform 4
 EBNA1BP2 EBNA1 binding protein 2
 HIST1H2AH Histone H2A type 1-H
 hCG_22804 hypothetical protein LOC645441
 NUCKS1 Isoform 1 of Nuclear ubiquitous casein and cyclin-dependent kinases substrate
 IGBP1 Immunoglobulin-binding protein 1
 ATPIF1 Putative uncharacterized protein DKFZp564G0422
 PCNP Isoform 1 of PEST proteolytic signal-containing nuclear protein
 RPS15 40S ribosomal protein S15
 - Similar to 60S ribosomal protein L35
 H2AFV Histone H2A.V
 CCDC124 Coiled-coil domain-containing protein 124
 RPS5 40S ribosomal protein S5
 BTF3 Isoform 1 of Transcription factor BTF3
 H1FX Histone H1x
 CNBP cDNA FLJ77718
 YBX1 Nuclease-sensitive element-binding protein 1
 NUPL1 Isoform 1 of Nucleoporin p58/p45
 MRPS33 MRPS33 protein (Fragment)
 RPL31 60S ribosomal protein L31
 FUS Isoform Short of RNA-binding protein FUS
 S100A4 Protein S100-A4
 PDIA3 14 kDa protein
 HINT1 Histidine triad nucleotide-binding protein 1
 EEF1A2 Elongation factor 1-alpha 2
 MRPL23 39S ribosomal protein L23, mitochondrial
 RPS26 Ribosomal protein 26 (RPS26) pseudogene
 PRPS1 Ribose-phosphate pyrophosphokinase 1
 MIF;LOC284889 Macrophage migration inhibitory factor
 PIN1 Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1
 EIF4B eukaryotic translation initiation factor 4B
 ENAH Isoform 2 of Protein enabled homolog
 CHMP4B Charged multivesicular body protein 4b
 UBQLN4 Ubiquilin-4
 CNPY2 Isoform 1 of Protein canopy homolog 2
 PGRMC1 Membrane-associated progesterone receptor component 1
 DBI Isoform a 1 of Acyl-CoA-binding protein
 TPD52L2 Isoform 2 of Tumor protein D54
 HIST1H1C Histone H1.2
 DRAP1 Isoform 1 of Dr1-associated corepressor
 TBCA Tubulin-specific chaperone A
 TIMM13 Mitochondrial import inner membrane translocase subunit Tim13
 RPL18 60S ribosomal protein L18
 TPD52 N8 protein long isoform (Fragment)
 MRPL49 39S ribosomal protein L49, mitochondrial
 UBE2N Ubiquitin-conjugating enzyme E2 N
 C14orf156 SRA stem-loop-interacting RNA-binding protein, mitochondrial
 FTL Ferritin light chain
 NDUFB10 NDUFB10 protein
 - 4 kDa protein
 HMGB2 High mobility group protein B2
 SFRS2 Splicing factor, arginine/serine-rich 2
 NAP1L4 Nucleosome assembly protein 1-like 4
 KHDRBS1 Isoform 1 of KH domain-containing, RNA-binding, signal transduction-associated
 PODXL2 Isoform 2 of Podocalyxin-like protein 2
 PSME1 Proteasome activator complex subunit 1
 HNRNPK Isoform 1 of Heterogeneous nuclear ribonucleoprotein K
 PSME2 Uncharacterized protein PSME2
 WHSC2 Isoform 1 of Negative elongation factor A
 EEF1E1 Eukaryotic translation elongation factor 1 epsilon-1
 FKBP3 FK506-binding protein 3
 RPS20 40S ribosomal protein S20
 NOLA3 H/ACA ribonucleoprotein complex subunit 3
 DDT D-dopachrome decarboxylase
 NAP1L1 Nucleosome assembly protein 1-like 1
 RPS24 Isoform 2 of 40S ribosomal protein S24
 CRMP1 collapsin response mediator protein 1 isoform 1
 RPS29 40S ribosomal protein S29
 BID Isoform 1 of BH3-interacting domain death agonist
  Protein Mascot Score  # of peptides  log2(heavy /light)  429
 195
 253
 1645
 625
 141
 194
 763
 1295
 320
 258
 340
 54
 241
 146
 2107
 68
 1110
 327
 421
 60
 286
 112
 130
 189
 210
 165
 130
 243
 155
 167
 1354
 49
 57
 230
 169
 115
 50
 288
 83
 100
 82
 163
 735
 450
 311
 82
 91
 77
 247
 223
 337
 115
 362
 144
 846
 111
 125
 479
 98
 143
 514
 154
 56
 221
 684
 258
 69
 177
 46
 581
 484
 275
 70
 48
 126
 178
 174
 91
 459
 249
 88
 47
 174
  9
 6
 5
 44
 17
 4
 4
 16
 34
 7
 6
 9
 1
 2
 2
 55
 1
 32
 8
 7
 1
 6
 3
 3
 4
 6
 5
 2
 5
 3
 4
 28
 1
 1
 5
 3
 3
 1
 7
 3
 2
 2
 3
 15
 10
 7
 2
 1
 2
 5
 6
 10
 1
 5
 2
 21
 2
 2
 10
 2
 4
 12
 3
 1
 6
 18
 8
 1
 4
 1
 12
 12
 8
 1
 1
 3
 6
 3
 2
 10
 5
 2
 1
 4
  -0.46
 -0.46
 -0.46
 -0.45
 -0.45
 -0.45
 -0.43
 -0.42
 -0.41
 -0.40
 -0.40
 -0.40
 -0.40
 -0.39
 -0.38
 -0.37
 -0.36
 -0.34
 -0.34
 -0.33
 -0.33
 -0.32
 -0.32
 -0.32
 -0.30
 -0.28
 -0.28
 -0.27
 -0.27
 -0.26
 -0.26
 -0.25
 -0.25
 -0.24
 -0.24
 -0.24
 -0.23
 -0.22
 -0.22
 -0.21
 -0.20
 -0.20
 -0.20
 -0.19
 -0.19
 -0.19
 -0.19
 -0.17
 -0.17
 -0.16
 -0.15
 -0.14
 -0.14
 -0.14
 -0.13
 -0.13
 -0.13
 -0.12
 -0.11
 -0.08
 -0.08
 -0.07
 -0.05
 -0.04
 -0.04
 -0.04
 -0.04
 -0.02
 0.00
 0.01
 0.01
 0.01
 0.02
 0.04
 0.04
 0.05
 0.05
 0.08
 0.08
 0.10
 0.11
 0.12
 0.12
 0.12
  87  Proteins eluted with mild detergent and salt (weak parkin interactors) RBP1 21 kDa protein
 SET Isoform 1 of Protein SET
 UBA52 ubiquitin and ribosomal protein L40 precursor
 S100A11 Protein S100-A11
 PFDN2 Prefoldin subunit 2
 KIAA1704 Isoform 3 of Uncharacterized protein KIAA1704
 C11orf59 UPF0404 protein C11orf59
 STX12 Syntaxin-12
 TUBB3 Tubulin beta-3 chain
 LOC100128936 similar to ribosomal protein L10a
 PAFAH1B3 Platelet-activating factor acetylhydrolase IB subunit gamma
 MYO15A Myosin-XV
 SOD1 Superoxide dismutase [Cu-Zn]
 NEDD8 Uncharacterized protein NEDD8 (Fragment)
 MRLC2 Myosin regulatory light chain
 NENF Neudesin
 PEF1 Peflin
 C19orf53 Leydig cell tumor 10 kDa protein homolog
 RBMX Heterogeneous nuclear ribonucleoprotein G
 RPL24 60S ribosomal protein L24
 RPS17 40S ribosomal protein S17
 - 28 kDa protein
 ATP6V1E1 Vacuolar proton pump subunit E 1
 HIST2H2BE Histone H2B type 2-E
 HIST2H4B;HIST1H4F;HIST1H4J;HIST1H4C;HIST1H4B;HIST1H4K;HIST1H4E;HIST2H4A;HIST1 H4H;HIST1H4L;HIST1H4A;HIST4H4;HIST1H4D;HIST1H4I Histone H4
 SNRPD2 Small nuclear ribonucleoprotein Sm D2
 ANP32A Acidic leucine-rich nuclear phosphoprotein 32 family member A
 TFRC Transferrin receptor protein 1
 SNRPG Small nuclear ribonucleoprotein G
 SCYE1 Multisynthetase complex auxiliary component p43
 LOC653314 similar to ribosomal protein L19
 EEF1B2 Elongation factor 1-beta
 CCT5 T-complex protein 1 subunit epsilon
 ATP5I ATP synthase, H+ transporting, mitochondrial F0 complex, subunit E
 TUBB2B Tubulin beta-2B chain
 PTBP1 Isoform 1 of Polypyrimidine tract-binding protein 1
 SNRPD1 Small nuclear ribonucleoprotein Sm D1
 - Uncharacterized protein ENSP00000348237
 AIP AH receptor-interacting protein
 STIP1 cDNA FLJ76863, highly similar to Homo sapiens stress-induced-phosphoprotein 1 (Hsp70/Hsp90-organizing protein) (STIP1), mRNA
 NPM1 nucleophosmin 1 isoform 3
 PRDX6 Peroxiredoxin-6
 HSPE1 10 kDa heat shock protein, mitochondrial
 DUT deoxyuridine triphosphatase isoform 1 precursor
 HNRNPA2B1 Isoform B1 of Heterogeneous nuclear ribonucleoproteins A2/B1
 FKBP4 FK506-binding protein 4
 GNB1 Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
 TUBA1A Tubulin alpha-1A chain
 - Uncharacterized protein ENSP00000375523
 HIST1H2BD Histone H2B type 1-D
 SORD Sorbitol dehydrogenase
 RPS21 40S ribosomal protein S21
 SYNCRIP Isoform 1 of Heterogeneous nuclear ribonucleoprotein Q
 SDHB Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial
 YWHAE 14-3-3 protein epsilon
 GOT2 Aspartate aminotransferase, mitochondrial
 GLO1 Lactoylglutathione lyase
 KHSRP Isoform 1 of Far upstream element-binding protein 2
 ACAT1 Acetyl-CoA acetyltransferase, mitochondrial
 SOD2 Superoxide dismutase [Mn], mitochondrial
 PDCD5 Programmed cell death protein 5
 SEC22B Vesicle-trafficking protein SEC22b
 CALM3;CALM2;CALM1 Calmodulin
 CKB Creatine kinase B-type
 PSMC1 26S protease regulatory subunit 4
 NME2 Nucleoside diphosphate kinase
 PGAM2 Phosphoglycerate mutase 2
 GSTP1 Glutathione S-transferase P
 TUBB Tubulin beta chain
 HSP90AB1 Heat shock protein HSP 90-beta
 HSPA9 Stress-70 protein, mitochondrial
 HMGA1 Isoform HMG-Y of High mobility group protein HMG-I/HMG-Y
 TPI1 Isoform 1 of Triosephosphate isomerase
 - Calnexin (Fragment)
 HSPA5 HSPA5 protein
 PPIA Peptidyl-prolyl cis-trans isomerase A
 PRDX2 Peroxiredoxin-2
 CALU Calumenin precursor
 TUBB4 Tubulin beta-4 chain
 CSTB Cystatin-B
 PRDX1 19 kDa protein
 RPS16 40S ribosomal protein S16
  Protein Mascot Score  # of peptides  log2(heavy /light)  416
 500
 103
 190
 464
 101
 157
 180
 296
 58
 51
 51
 1774
 239
 79
 78
 163
 76
 156
 74
 450
 257
 472
 78
  8
 11
 3
 3
 13
 3
 2
 5
 7
 1
 1
 3
 38
 6
 2
 1
 2
 2
 3
 1
 11
 5
 7
 2
  0.13
 0.13
 0.14
 0.16
 0.17
 0.18
 0.18
 0.20
 0.24
 0.24
 0.25
 0.26
 0.27
 0.28
 0.29
 0.29
 0.30
 0.30
 0.30
 0.30
 0.31
 0.33
 0.34
 0.35
  778
 63
 964
 55
 110
 401
 51
 80
 168
 69
 182
 71
 352
 247
 94
  16
 1
 24
 1
 2
 11
 1
 1
 4
 2
 2
 1
 7
 7
 2
  0.36
 0.36
 0.37
 0.37
 0.37
 0.41
 0.41
 0.42
 0.43
 0.44
 0.45
 0.46
 0.46
 0.48
 0.48
  351
 1768
 287
 1436
 120
 124
 58
 48
 1210
 180
 790
 76
 137
 84
 132
 650
 62
 63
 257
 531
 111
 535
 73
 2025
 1382
 193
 314
 103
 260
 1950
 186
 707
 57
 836
 167
 159
 2425
 222
 126
 63
 61
 197
 60
  9
 30
 5
 30
 2
 3
 1
 1
 26
 3
 19
 2
 4
 2
 3
 16
 2
 1
 6
 14
 2
 11
 1
 43
 27
 4
 10
 2
 4
 42
 5
 15
 1
 18
 2
 3
 52
 5
 3
 1
 1
 5
 1
  0.50
 0.51
 0.52
 0.52
 0.52
 0.54
 0.54
 0.56
 0.56
 0.58
 0.60
 0.62
 0.62
 0.62
 0.63
 0.65
 0.66
 0.67
 0.67
 0.70
 0.72
 0.72
 0.75
 0.75
 0.76
 0.76
 0.76
 0.76
 0.80
 0.80
 0.80
 0.81
 0.81
 0.83
 0.84
 0.85
 0.86
 0.86
 0.88
 0.88
 0.88
 0.89
 0.89
  88  Proteins eluted with mild detergent and salt (weak parkin interactors) EEF1AL3;EEF1A1 Putative elongation factor 1-alpha-like 3
 PFN1 Profilin-1
 TOMM34 Mitochondrial import receptor subunit TOM34
 - Peptidyl-prolyl cis-trans isomerase
 NASP Isoform 1 of Nuclear autoantigenic sperm protein
 YWHAG 14-3-3 protein gamma
 CCT2 T-complex protein 1 subunit beta
 ACTB Actin, cytoplasmic 1
 EEF2 Elongation factor 2
 PDIA3 Protein disulfide-isomerase A3
 HSPD1 60 kDa heat shock protein, mitochondrial
 EFTUD2 116 kDa U5 small nuclear ribonucleoprotein component
 HSPA8 Isoform 1 of Heat shock cognate 71 kDa protein
 PSMC5 26S protease regulatory subunit 8
 KPNB1 Importin subunit beta-1
 PDIA6 Isoform 2 of Protein disulfide-isomerase A6
 GPI Glucose-6-phosphate isomerase
 CAPZA1 F-actin-capping protein subunit alpha-1
 PCMT1 Isoform 1 of Protein-L-isoaspartate(D-aspartate) O-methyltransferase
 CCT6A T-complex protein 1 subunit zeta
 PRDX3 Thioredoxin-dependent peroxide reductase, mitochondrial
 hCG_1984468 hypothetical protein LOC389672
 YWHAB Isoform Long of 14-3-3 protein beta/alpha
 MDH2 Malate dehydrogenase, mitochondrial
 HSPH1 Heat shock 105kDa/110kDa protein 1
 CFL1 Cofilin-1
 ENO1 Isoform alpha-enolase of Alpha-enolase
 GAPDH Glyceraldehyde-3-phosphate dehydrogenase
 ALCAM Isoform 1 of CD166 antigen
 ATP5B ATP synthase subunit beta, mitochondrial
 CLIC1 Chloride intracellular channel protein 1
 AK1 Adenylate kinase isoenzyme 1
 FARSA Phenylalanyl-tRNA synthetase alpha chain
 SSB Lupus La protein
 DCI Isoform 1 of 3,2-trans-enoyl-CoA isomerase, mitochondrial
 SNRPD3 Small nuclear ribonucleoprotein Sm D3
 NCL Isoform 1 of Nucleolin
 - Uncharacterized protein ENSP00000382131 (Fragment)
 MSI2 Isoform 1 of RNA-binding protein Musashi homolog 2
 PSMA5 Proteasome subunit alpha type-5
 PIR Pirin
 ENO2 Gamma-enolase
 PKM2 Isoform M2 of Pyruvate kinase isozymes M1/M2
 PGK1 Phosphoglycerate kinase 1
 NP CDNA FLJ25678 fis, clone TST04067, highly similar to PURINE NUCLEOSIDE PHOSPHORYLASE
 ANXA5 Annexin A5
 PYCR1 pyrroline-5-carboxylate reductase 1 isoform 2
 LOC439992 similar to v-fos transformation effector protein isoform 2
 ATP5A1 ATP synthase subunit alpha, mitochondrial
 ALDOA Fructose-bisphosphate aldolase A
 NCAM1 Isoform 3 of Neural cell adhesion molecule 1
 SNX13 Isoform 2 of Sorting nexin-13
  Protein Mascot Score  # of peptides  log2(heavy /light)  331
 891
 49
 31
 57
 207
 110
 1085
 151
 349
 1780
 48
 179
 54
 264
 334
 200
 52
 238
 81
 319
 51
 83
 644
 54
 296
 2995
 1378
 53
 238
 83
 472
 99
 320
 55
 48
 328
 22
 73
 134
 133
 199
 1001
 711
  11
 18
 1
 1
 1
 3
 3
 25
 3
 9
 43
 1
 5
 1
 4
 6
 4
 1
 8
 2
 6
 1
 1
 11
 1
 8
 56
 32
 1
 6
 2
 11
 2
 6
 1
 1
 8
 1
 2
 2
 2
 3
 22
 13
  0.89
 0.90
 0.93
 0.93
 0.94
 0.95
 0.95
 0.95
 0.97
 0.97
 0.99
 0.99
 0.99
 1.00
 1.00
 1.01
 1.01
 1.02
 1.02
 1.02
 1.03
 1.03
 1.04
 1.04
 1.05
 1.06
 1.06
 1.07
 1.07
 1.07
 1.09
 1.10
 1.10
 1.11
 1.12
 1.21
 1.21
 1.22
 1.23
 1.24
 1.31
 1.34
 1.39
 1.42
  50
 174
 89
 86
 274
 59
 48
 46
  1
 2
 2
 1
 5
 1
 1
 1
  1.48
 1.50
 1.50
 1.56
 1.58
 1.59
 2.01
 4.44
  89  Table 4. FLAG-parkin pull-down proteins with SILAC ratios – strong interactor fraction Proteins eluted with SDS sample buffer (strong parkin interactors) SKP1 Isoform 2 of S-phase kinase-associated protein 1 IVNS1ABP Influenza virus NS1A-binding protein
 NAPA Alpha-soluble NSF attachment protein
 PPA1 Inorganic pyrophosphatase
 CDC37 Hsp90 co-chaperone Cdc37
 FBXO21 FBXO21 protein
 PARK7 Protein DJ-1
 HNRNPD Isoform 1 of Heterogeneous nuclear ribonucleoprotein D0
 DUT Isoform DUT-M of Deoxyuridine 5'-triphosphate nucleotidohydrolase, mitochondrial
 ENO3 Beta-enolase
 ENO2 Gamma-enolase
 ATP6V1G2 Vacuolar proton pump subunit G 2
 PDIA3 14 kDa protein
 - 28 kDa protein
 NEFH Neurofilament heavy polypeptide
 SPIN3 Isoform 1 of Spindlin-3
 PRDX1 Peroxiredoxin-1
 KRT9 Keratin, type I cytoskeletal 9
 HNRNPA1 Isoform A1-B of Heterogeneous nuclear ribonucleoprotein A1
 CALR Calreticulin
 PSMC4 Isoform 1 of 26S protease regulatory subunit 6B
 TUBA1B Tubulin alpha-1B chain
 UBA52 ubiquitin and ribosomal protein L40 precursor
 LOC646817 similar to template acyivating factor-I alpha
 PDIA3 Protein disulfide-isomerase A3
 EIF3F Eukaryotic translation initiation factor 3 subunit 5
 KIAA0515 hypothetical protein LOC84726
 TUBA1A Tubulin alpha-1A chain
 PRDX2 Peroxiredoxin-2
 ACTA2 Actin, aortic smooth muscle
 HSPA5 HSPA5 protein
 CRKL Crk-like protein
 STIP1 Stress-induced-phosphoprotein 1
 ARHGDIA Rho GDP-dissociation inhibitor 1
 TPM3 Putative uncharacterized protein DKFZp686J1372
 TAGLN2 Transgelin-2
 RBP1 Retinol-binding protein I, cellular
 TUBB2A Tubulin beta-2A chain
 GTF2I Isoform 1 of General transcription factor II-I
 HSP90AB1 Heat shock protein HSP 90-beta
 UCHL1 Ubiquitin carboxyl-terminal hydrolase isozyme L1
 PEBP1 Phosphatidylethanolamine-binding protein 1
 - RcNSEP1 (Fragment)
 YWHAE 14-3-3 protein epsilon
 YWHAQ 14-3-3 protein theta
 BOLA2;BOLA2B Isoform 2 of BolA-like protein 2
 ACTB Actin, cytoplasmic 1
 HNRNPK Heterogeneous nuclear ribonucleoprotein K
 PRDX4 Peroxiredoxin-4
 TUBB Tubulin beta chain
 HSPA8 Isoform 1 of Heat shock cognate 71 kDa protein
 PFDN5 Prefoldin subunit 5
 CFL1 Cofilin-1
 NCL cDNA FLJ45706 fis, clone FEBRA2028457, highly similar to Nucleolin
 GNB1 Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
 YWHAG 14-3-3 protein gamma
 HSP90AA1 Isoform 1 of Heat shock protein HSP 90-alpha
 ATP5B ATP synthase subunit beta, mitochondrial
 NPM1 Isoform 1 of Nucleophosmin
 ATP5A1 ATP synthase subunit alpha, mitochondrial
 PPIA Peptidyl-prolyl cis-trans isomerase A
 HSPD1 60 kDa heat shock protein, mitochondrial
 CANX Calnexin
 RCN2 Reticulocalbin-2
 CALU Calumenin precursor
 TUBB2C Tubulin beta-2C chain
 ACTG1 Actin, cytoplasmic 2
 HSPA9 Stress-70 protein, mitochondrial
 EIF5A2 Eukaryotic translation initiation factor 5A-2
 TCEB2 Transcription elongation factor B polypeptide 2
 UCHL3 Ubiquitin carboxyl-terminal hydrolase isozyme L3
 PFN1 Profilin-1
 MAP3K7IP1 Mitogen-activated protein kinase kinase kinase 7-interacting protein 1
 CCT2 T-complex protein 1 subunit beta
 EEF1B2 Elongation factor 1-beta
 HNRNPL heterogeneous nuclear ribonucleoprotein L isoform a
 WDR77 Methylosome protein 50
 CSTB Cystatin-B
 PGK1 Phosphoglycerate kinase 1
 LOC100133841 similar to Peptidase D
  Protein Mascot Score  # of peptides  log2(heavy /light)  72 231 92 187 55
 54
 52
 45
 395
 68
 172
 148
 69
 303
 97
 51
 552
 351
 287
 142
 69
 58
 134
 298
 401
 274
 45
 2949
 148
 21
 287
 101
 181
 425
 357
 86
 363
 332
 58
 1324
 491
 608
 121
 541
 183
 57
 338
 493
 208
 3864
 1891
 96
 1091
 586
 63
 613
 337
 306
 187
 48
 1985
 2143
 131
 149
 282
 78
 1178
 685
 138
 107
 72
 932
 53
 148
 66
 46
 518
 280
 50
 74
  1
 4
 3
 4
 1
 1
 1
 1
 5
 1
 3
 3
 1
 5
 2
 1
 13
 6
 4
 3
 1
 1
 3
 5
 9
 2
 1
 51
 4
 1
 6
 2
 2
 8
 7
 2
 7
 4
 1
 36
 14
 10
 2
 11
 3
 1
 5
 12
 4
 69
 40
 2
 22
 10
 1
 10
 8
 5
 4
 1
 38
 43
 2
 3
 8
 3
 21
 16
 2
 2
 2
 18
 1
 3
 1
 1
 8
 8
 1
 1
  -1.42 -1.25
 -1.20
 -1.13
 -1.04
 -0.89
 -0.83
 -0.83
 -0.76
 -0.71
 -0.61
 -0.60
 -0.57
 -0.56
 -0.54
 -0.51
 -0.50
 -0.49
 -0.48
 -0.48
 -0.44
 -0.44
 -0.44
 -0.42
 -0.34
 -0.31
 -0.31
 -0.30
 -0.30
 -0.29
 -0.28
 -0.28
 -0.28
 -0.28
 -0.27
 -0.27
 -0.27
 -0.25
 -0.23
 -0.22
 -0.20
 -0.20
 -0.17
 -0.17
 -0.17
 -0.11
 -0.10
 -0.10
 -0.10
 -0.09
 -0.09
 -0.08
 -0.08
 -0.08
 -0.07
 -0.06
 -0.06
 -0.05
 -0.05
 -0.04
 -0.03
 -0.03
 -0.03
 0.01
 0.01
 0.02
 0.02
 0.05
 0.06
 0.06
 0.15
 0.17
 0.17
 0.17
 0.21
 0.22
 0.24
 0.27
 0.28
 0.29
  90  Proteins eluted with SDS sample buffer (strong parkin interactors) CKB Creatine kinase B-type
 HNRNPA2B1 Isoform B1 of Heterogeneous nuclear ribonucleoproteins A2/B1
 PRDX5 Isoform Mitochondrial of Peroxiredoxin-5, mitochondrial
 GSTP1 Glutathione S-transferase P
 HSPE1 10 kDa heat shock protein, mitochondrial
 YWHAB Isoform Long of 14-3-3 protein beta/alpha
 PFN2 Isoform IIb of Profilin-2
 HNRNPH1 Heterogeneous nuclear ribonucleoprotein H
 PA2G4 20 kDa protein
 ECH1 Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase, mitochondrial
 ENO1 Isoform alpha-enolase of Alpha-enolase
 TUBB3 Tubulin beta-3 chain
 TPI1 Isoform 1 of Triosephosphate isomerase
 MIF;LOC284889 Macrophage migration inhibitory factor
 ATAD4 Proteasome 26S ATPase subunit 5 variant (Fragment)
 NP CDNA FLJ25678 fis, clone TST04067, highly similar to PURINE NUCLEOSIDE PHOSPHORYLASE
 PDIA6 Isoform 2 of Protein disulfide-isomerase A6
 DPYSL2 Dihydropyrimidinase-related protein 2
 PCMT1 Isoform 1 of Protein-L-isoaspartate(D-aspartate) O-methyltransferase
 STRAP Serine-threonine kinase receptor-associated protein
 MDH2 Malate dehydrogenase, mitochondrial
 PRDX6 Peroxiredoxin-6
 NME1;NME2 Nucleoside diphosphate kinase A
 CCT8 59 kDa protein
 PRMT5 protein arginine methyltransferase 5 isoform b
 PAFAH1B3 Platelet-activating factor acetylhydrolase IB subunit gamma
 FKBP4 FK506-binding protein 4
 ACAT1 Acetyl-CoA acetyltransferase, mitochondrial
 ALDOA Fructose-bisphosphate aldolase A
 PKM2 Isoform M1 of Pyruvate kinase isozymes M1/M2
 GAPDH Glyceraldehyde-3-phosphate dehydrogenase
  Protein Mascot Score  # of peptides  log2(heavy /light)  683
 144
 49
 329
 243
 235
 120
 104
 76
 127
 1159
 430
 239
 174
 88
  14
 4
 1
 4
 3
 4
 2
 3
 2
 2
 21
 7
 5
 3
 1
  0.29
 0.30
 0.30
 0.32
 0.34
 0.38
 0.39
 0.40
 0.42
 0.45
 0.46
 0.48
 0.53
 0.55
 0.56
  54
 53
 110
 580
 119
 83
 259
 107
 60
 124
 53
 57
 194
 112
 124
 448
  1
 1
 2
 11
 2
 2
 6
 3
 1
 3
 1
 1
 5
 3
 2
 10
  0.58
 0.59
 0.59
 0.59
 0.60
 0.61
 0.62
 0.72
 0.86
 0.88
 0.92
 0.98
 1.02
 1.15
 1.33
 1.44
  91  Table 5. FLAG-parkin pull-down proteins under CCCP with SILAC ratios – strong interactor fraction Proteins eluted with SDS sample buffer (strong parkin interactors – CCCP conditions)  Protein Mascot Score  # of peptides  log2(heavy /light)  KIAA1618 Isoform 3 of Protein ALO17 SH3YL1 isoform 5 of SH3 domain-containing YSC84-like protein 1 MRLC2 Myosin regulatory light chain
 TMEM64 transmembrane protein 64 PSMC6 26S protease regulatory subunit S10B MYH9 Myosin-9
 EIF4A1 Eukaryotic initiation factor 4A-I
 AIFM1 Isoform 1 of Apoptosis-inducing factor 1, mitochondrial RAD50 Isoform 1 of DNA repair protein RAD50
 MYL6;MYL6B Isoform Non-muscle of Myosin light polypeptide 6
 HYOU1 Hypoxia up-regulated protein 1
 RPS24 Isoform 1 of 40S ribosomal protein S24
 CALU Isoform 2 of Calumenin
 MYH10 Isoform 1 of Myosin-10
 AKR1C1 Aldo-keto reductase family 1 member C1
 UCHL5 Isoform 2 of Ubiquitin carboxyl-terminal hydrolase isozyme L5
 SF1 Isoform 2 of Splicing factor 1
 GTF2F2 General transcription factor IIF subunit 2
 LOC728453 similar to 40S ribosomal protein S28
 EIF3E Eukaryotic translation initiation factor 3 subunit E
 CALR Calreticulin
 DBN1 Isoform 1 of Drebrin
 TMOD2 Tropomodulin-2
 CAPZB Capping protein
 LIMA1 Isoform Beta of LIM domain and actin-binding protein 1
 PDIA3 Protein disulfide-isomerase A3
 RPS28 40S ribosomal protein S28
 FOLH1 Isoform PSMA-1 of Glutamate carboxypeptidase 2
 ATAD3B TOB3
 ACTB Actin, cytoplasmic 1
 ABLIM1 Isoform 1 of Actin-binding LIM protein 1
 CAPZA1 F-actin-capping protein subunit alpha-1
 CCNT1 Cyclin-T1
 DHX15 Putative pre-mRNA-splicing factor ATP-dependent RNA helicase DHX15
 ACTG1 Actin, cytoplasmic 2
 PFKFB3 Fructose-6-phosphate,2-kinase/fructose-2, 6-bisphosphatase (Fragment)
 L1TD1 LINE-1 type transposase domain-containing protein 1
 SVIL Isoform 2 of Supervillin
 SLC9A5 Solute carrier family 9 (Sodium/hydrogen exchanger), isoform 5 variant
 ACTA2 Actin, aortic smooth muscle
 TPM4 Isoform 1 of Tropomyosin alpha-4 chain
 HDX Isoform 2 of Highly divergent homeobox
 IDH2 Isocitrate dehydrogenase [NADP], mitochondrial
 CHGB Secretogranin-1
 EIF3I Eukaryotic translation initiation factor 3 subunit I
 EXOSC8 Exosome complex exonuclease RRP43
 HSPA5 HSPA5 protein
 KLKB1 Plasma kallikrein
 EIF3F Eukaryotic translation initiation factor 3 subunit 5
 SEPT11 Septin-11
 SUMO3 Small ubiquitin-related modifier 3
 RPL8 60S ribosomal protein L8
 CCDC39 Coiled-coil domain-containing protein 39
 GPR78 Probable G-protein coupled receptor 78
 NBN Nibrin
 ACTR1A Alpha-centractin
 EIF4B eukaryotic translation initiation factor 4B
 UBA52 ubiquitin and ribosomal protein L40 precursor
 RPL4 60S ribosomal protein L4
 RBM17 Splicing factor 45
 TPM3 Putative uncharacterized protein DKFZp686J1372
 SIP1 Isoform 1 of Survival of motor neuron protein-interacting protein 1
 RPL29P4;RPL29 Novel protein similar to ribosomal protein L29 RPL29
 LSM14A Isoform 2 of LSM14 protein homolog A
 CEP170 Isoform 1 of Centrosomal protein of 170 kDa
 TBR1 T-brain-1 protein
 EPB41L3 Isoform A of Band 4.1-like protein 3
 RPL13A 60S ribosomal protein L13a
 RPS3 40S ribosomal protein S3
 S100A6 Protein S100-A6
 RPS20 40S ribosomal protein S20
 VCP Transitional endoplasmic reticulum ATPase
  33
 28
 700
 26 40 1722
 101
 52
 105
 187
 133
 254
 287
 995
 25
 57
 188
 30
 32
 32
 28
 26
 35
 223
 2024
 34
 345
 182
 429
 79
 767
 66
 27
 29
 1019
 146
 281
 50
 163
 12531
 33
 26
 66
 42
 222
 327
 442
 41
 79
 378
 510
 1507
 25
 330
 64
 93
 374
 26
 26
 28
 144
 452
 665
 29
 90
 430
 44
 59
 180
 78
 41
 88
  1 1 13 1 1 32 3 1 3 4 3 4 7 19 2 1 1 1 1 1 4 33 3 9 1 17 2 1 1 14 3 7 2 4 178 1
 1
 2
 1
 5
 6
 7
 1
 2
 8
 9
 26
 1
 3
 2
 2
 8
 1
 1
 1
 3
 11
 11
 1
 2
 5
 1
 1
 3
 1
 1
 1
 1
 2
 1
 3
 4
  6.29 3.60 19.14 3.49 2.68 16.95
 1.88
 1.88
 1.73
 1.61
 1.56
 1.46
 1.44
 1.57
 1.36
 1.35
 1.35
 1.30
 1.24
 1.23
 1.19
 1.17
 1.13
 1.09
 1.08
 1.07
 1.06
 1.05
 1.05
 1.05
 1.04
 1.04
 1.04
 1.01
 1.00
 0.99
 0.96
 0.95
 0.95
 0.93
 0.92
 0.90
 0.89
 0.88
 0.86
 0.85
 0.85
 0.82
 0.81
 0.79
 0.77
 0.76
 0.75
 0.74
 0.71
 0.71
 0.70
 0.70
 0.69
 0.69
 0.69
 0.67
 0.67
 0.66
 0.66
 0.64
 0.64
 0.62
 0.62
 0.62
 0.62
 0.62
  92  Proteins eluted with SDS sample buffer (strong parkin interactors – CCCP conditions) PPP2R1A Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform
 KCTD5 BTB/POZ domain-containing protein KCTD5
 HSP90AA1 Isoform 1 of Heat shock protein HSP 90-alpha
 CSTB Cystatin-B
 RPL9 60S ribosomal protein L9
 IMMT Isoform 1 of Mitochondrial inner membrane protein
 FUBP1 Isoform 1 of Far upstream element-binding protein 1
 PSMC3 26S protease regulatory subunit 6A
 PSMD11 Proteasome 26S non-ATPase subunit 11 variant (Fragment)
 PPIB peptidylprolyl isomerase B precursor
 CALD1 Isoform 3 of Caldesmon
 LOC643677 similar to hCG2011852
 FSCN1 Fascin
 VPS72 Vacuolar protein sorting-associated protein 72 homolog
 HSP90AB1 Heat shock protein HSP 90-beta
 HSPA1L Heat shock 70 kDa protein 1L
 GSR Isoform Mitochondrial of Glutathione reductase, mitochondrial
 CCAR1 Cell division cycle and apoptosis regulator protein 1
 VGF Neurosecretory protein VGF
 TUFM Tu translation elongation factor, mitochondrial precursor
 TXN Thioredoxin
 RPL14 60S ribosomal protein L14
 CLASP2 CLASP2 protein
 CTTN Src substrate cortactin
 PDCD6 Programmed cell death protein 6
 RPS14 40S ribosomal protein S14
 PFDN2 Prefoldin subunit 2
 CRMP1 Dihydropyrimidinase-related protein 1
 UBE2N Ubiquitin-conjugating enzyme E2 N
 ATP5B ATP synthase subunit beta, mitochondrial
 MTDH Protein LYRIC
 RPL36AL 60S ribosomal protein L36a-like
 PSMD4 Isoform Rpn10A of 26S proteasome non-ATPase regulatory subunit 4
 DUT Isoform DUT-M of Deoxyuridine 5'-triphosphate nucleotidohydrolase, mitochondrial
 DCTN2 6 kDa protein
 YWHAQ 14-3-3 protein theta
 C1orf57 Probable UPF0334 kinase-like protein C1orf57
 PHB2 Prohibitin-2
 HSPD1 60 kDa heat shock protein, mitochondrial
 PPP1R9B protein phosphatase 1, regulatory subunit 9B
 THOC4 THO complex subunit 4
 RPS8 40S ribosomal protein S8
 HNRNPH3 Isoform 1 of Heterogeneous nuclear ribonucleoprotein H3
 PDIA6 Isoform 2 of Protein disulfide-isomerase A6
 ZNF415 Isoform 1 of Zinc finger protein 415
 STARD13 Isoform 2 of StAR-related lipid transfer protein 13
 HIGD1B 10 kDa protein
 LRRFIP1 Isoform 2 of Leucine-rich repeat flightless-interacting protein 1
 MAP1S Microtubule-associated protein 1S
 RBM14 Isoform 1 of RNA-binding protein 14
 PPP1CC Isoform Gamma-1 of Serine/threonine-protein phosphatase PP1-gamma catalytic subunit
 RPS19 40S ribosomal protein S19
 LOC284064 similar to ribosomal protein L29
 VGF VGF nerve growth factor inducible precursor
 EEF1G Elongation factor 1-gamma
 RPL7 60S ribosomal protein L7
 SCG2 Secretogranin-2
 RPL35A 60S ribosomal protein L35a
 SGTA Small glutamine-rich tetratricopeptide repeat-containing protein alpha
 PSMC1 26S protease regulatory subunit 4
 SCYE1 Multisynthetase complex auxiliary component p43
 PFN2 Isoform IIb of Profilin-2
 CCT5 T-complex protein 1 subunit epsilon
 SNRPG Small nuclear ribonucleoprotein G
 HSPA8 Isoform 1 of Heat shock cognate 71 kDa protein
 CORO1C Coronin-1C_i3 protein
 RPSA 33 kDa protein
 EEF1D Elongation factor 1-delta
 - 15 kDa protein
 RPLP0 60S acidic ribosomal protein P0
 HAGH hydroxyacyl glutathione hydrolase isoform 1
 KPNB1 Importin subunit beta-1
 PSMC2 26S protease regulatory subunit 7
 SLC25A5 ADP/ATP translocase 2
 RPS3A 40S ribosomal protein S3a
 EMID1 Isoform 1 of EMI domain-containing protein 1
 CCT8 59 kDa protein
 FAM40A Isoform 3 of Protein FAM40A
 NEFL Neurofilament light polypeptide
 DARS Aspartyl-tRNA synthetase, cytoplasmic
 ACAT1 Acetyl-CoA acetyltransferase, mitochondrial
  Protein Mascot Score  # of peptides  log2(heavy /light)  31
 110
 37
 121
 281
 154
 73
 449
 46
 55
 106
 58
 171
 289
 162
 212
 25
 100
 28
 1015
 120
 106
 40
 189
 478
 83
 47
 33
 31
 92
 181
 107
 32
 151
 733
 32
 25
 1852
 257
 56
 541
 30
 60
 1675
 183
 428
 187
 97
 84
 64
 26
 27
 79
 32
 148
 44
 134
 28
 26
 180
 56
 47
 70
 85
 338
 165
 130
 102
 34
 3605
 115
 1335
 225
 42
 570
 32
 39
 365
 465
 92
 27
  3
 1
 8
 1
 1
 2
 2
 3
 5
 3
 4
 1
 3
 1
 17
 3
 3
 1
 4
 8
 1
 1
 1
 1
 3
 3
 3
 1
 3
 14
 1
 1
 29
 6
 1
 8
 1
 1
 24
 4
 7
 4
 2
 1
 2
 1
 1
 2
 1
 3
 1
 3
 1
 1
 3
 2
 1
 2
 2
 6
 4
 2
 2
 1
 57
 3
 16
 3
 1
 11
 1
 1
 7
 8
 2
 1
 4
 1
 12
 3
 26
  0.59
 0.59
 0.59
 0.58
 0.58
 0.57
 0.57
 0.57
 0.57
 0.56
 0.55
 0.55
 0.53
 0.52
 0.52
 0.50
 0.49
 0.49
 0.49
 0.49
 0.48
 0.48
 0.46
 0.46
 0.46
 0.46
 0.43
 0.43
 0.43
 0.41
 0.39
 0.39
 0.39
 0.39
 0.38
 0.38
 0.38
 0.37
 0.36
 0.36
 0.36
 0.35
 0.35
 0.35
 0.34
 0.34
 0.33
 0.33
 0.32
 0.32
 0.32
 0.32
 0.31
 0.31
 0.30
 0.30
 0.29
 0.29
 0.29
 0.29
 0.29
 0.29
 0.28
 0.28
 0.27
 0.27
 0.27
 0.27
 0.26
 0.26
 0.25
 0.25
 0.25
 0.24
 0.24
 0.24
 0.24
 0.23
 0.23
 0.23
 0.22
  93  Proteins eluted with SDS sample buffer (strong parkin interactors – CCCP conditions) RPL3 60S ribosomal protein L3
 YBX1 Nuclease-sensitive element-binding protein 1
 HNRNPA1 Isoform A1-B of Heterogeneous nuclear ribonucleoprotein A1
 TUBB2B Tubulin beta-2B chain
 RPL12 Isoform 1 of 60S ribosomal protein L12
 LOC388344 similar to RPL13 protein
 - Calnexin (Fragment)
 PPM1B Isoform Beta-1 of Protein phosphatase 1B
 CGNL1 Isoform 1 of Cingulin-like protein 1
 RPL28 60S ribosomal protein L28
 MATR3 Matrin-3
 FLJ12529 Isoform 1 of Cleavage and polyadenylation specificity factor subunit 7
 SLC25A3 Isoform A of Phosphate carrier protein, mitochondrial
 FHL1 Four and a half LIM domains 1 variant
 MAP1B Microtubule-associated protein 1B
 PSMC5 26S protease regulatory subunit 8
 PSMC4 Isoform 1 of 26S protease regulatory subunit 6B
 NME2 Nucleoside diphosphate kinase
 TUBB1 Tubulin beta-1 chain
 TMPO Isoform Gamma of Lamina-associated polypeptide 2, isoforms beta/gamma
 YWHAH 14-3-3 protein eta
 ATP5A1 ATP synthase subunit alpha, mitochondrial
 IFT74 Intraflagellar transport protein 74 homolog
 CFL1 Cofilin-1
 PKM2 Isoform M2 of Pyruvate kinase isozymes M1/M2
 PRDX3 Thioredoxin-dependent peroxide reductase, mitochondrial
 YWHAG 14-3-3 protein gamma
 STMN2 Stathmin-2
 PCBP2 poly(rC) binding protein 2 isoform b
 VIM Vimentin
 C22orf28 UPF0027 protein C22orf28
 SERBP1 Isoform 1 of Plasminogen activator inhibitor 1 RNA-binding protein
 ACTL8 Actin-like protein 8
 EWSR1 cDNA FLJ31747 fis, clone NT2RI2007377, highly similar to RNA-BINDING PROTEIN 
 CLTC Isoform 1 of Clathrin heavy chain 1
 MRPL12 39S ribosomal protein L12, mitochondrial
 RPL5 60S ribosomal protein L5
 MUTED;TXNDC5 Thioredoxin domain-containing protein 5
 EEF1D eukaryotic translation elongation factor 1 delta isoform 1
 HNRNPK Isoform 1 of Heterogeneous nuclear ribonucleoprotein K
 EEF1B2 Elongation factor 1-beta
 IGF2BP1 Insulin-like growth factor 2 mRNA-binding protein 1
 SEPT7 Isoform 1 of Septin-7
 SPTAN1 Isoform 2 of Spectrin alpha chain, brain
 ILF2 Interleukin enhancer-binding factor 2
 WDR77 Methylosome protein 50
 PCMT1 Isoform 1 of Protein-L-isoaspartate(D-aspartate) O-methyltransferase
 SLAIN2 SLAIN motif-containing protein 2
 TUBB2C Tubulin beta-2C chain
 PSMD2 26S proteasome non-ATPase regulatory subunit 2
 DNAJA2 DnaJ homolog subfamily A member 2
 ARHGDIA Rho GDP-dissociation inhibitor 1
 MAP2 Isoform 1 of Microtubule-associated protein 2
 HIST2H4B;HIST1H4F;HIST1H4J;HIST1H4C;HIST1H4B;HIST1H4K;HIST1H4E;HIST2H4A;HIST1 H4H;HIST1H4L;HIST1H4A;HIST4H4;HIST1H4D;HIST1H4I Histone H4
 PHB Prohibitin
 ARGLU1 Isoform 1 of Arginine and glutamate-rich protein 1
 ACTN4 Alpha-actinin-4
 POLR2E DNA-directed RNA polymerases I, II, and III subunit RPABC1
 HDGF Hepatoma-derived growth factor
 PRDX4 Peroxiredoxin-4
 YWHAE 14-3-3 protein epsilon
 HIST1H1C Histone H1.2
 KLC1 kinesin light chain 1 isoform 2
 NUDT21 Cleavage and polyadenylation specificity factor subunit 5
 SUB1 Activated RNA polymerase II transcriptional coactivator p15
 RPL23A;hCG_16001 60S ribosomal protein L23a
 HNRNPR Heterogeneous nuclear ribonucleoprotein R
 INA Alpha-internexin
 CCT2 T-complex protein 1 subunit beta
 DYNC1I2 Isoform 2D of Cytoplasmic dynein 1 intermediate chain 2
 CCT7 T-complex protein 1 subunit eta
 SF3A3 Splicing factor 3A subunit 3
 RPL11 Isoform 1 of 60S ribosomal protein L11
 PRMT5 Protein arginine N-methyltransferase 5
 HDLBP cDNA FLJ45936 fis, clone PLACE7004103, highly similar to Vigilin
 TCP1 T-complex protein 1 subunit alpha
 SNRPC U1 small nuclear ribonucleoprotein C
 SF3B3 Isoform 1 of Splicing factor 3B subunit 3
 NME1;NME2 Nucleoside diphosphate kinase A
 GNB1 Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
  Protein Mascot Score  # of peptides  log2(heavy /light)  215
 25
 755
 143
 1757
 197
 795
 257
 432
 648
 75
 28
 1241
 33
 80
 84
 93
 100
 28
 9042
 79
 620
 25
 85
 239
 38
 1260
 44
 750
 1752
 370
 647
 97
 65
 1479
 64
 31
 34
 880
 418
 79
 125
 129
 45
 1334
 449
 178
 102
 1075
 56
 3106
 898
 60
  3
 10
 5
 7
 8
 1
 1
 19
 1
 2
 2
 2
 2
 1
 148
 2
 13
 1
 2
 5
 1
 19
 1
 10
 26
 4
 11
 2
 1
 28
 2
 1
 1
 14
 8
 2
 3
 2
 1
 22
 4
 3
 3
 25
 1
 52
 16
 1
 18
 9
 4
 6
 4
  0.22
 0.22
 0.22
 0.21
 0.21
 0.21
 0.21
 0.21
 0.21
 0.21
 0.20
 0.20
 0.20
 0.20
 0.20
 0.19
 0.18
 0.18
 0.18
 0.17
 0.17
 0.17
 0.17
 0.16
 0.15
 0.14
 0.14
 0.13
 0.13
 0.12
 0.12
 0.12
 0.12
 0.11
 0.11
 0.10
 0.10
 0.10
 0.10
 0.10
 0.09
 0.09
 0.09
 0.08
 0.08
 0.08
 0.07
 0.07
 0.07
 0.07
 0.06
 0.06
 0.06
  1285
 487
 137
 262
 179
 138
 98
 168
 83
 54
 39
 58
 198
 48
 75
 150
 29
 50
 88
 1468
 412
 323
 145
 64
 152
 3221
 40
  3
 2
 5
 2
 1
 1
 1
 3
 1
 2
 3
 1
 1
 2
 25
 8
 4
 3
 1
 4
 64
 1
 10
 1
 3
 6
 3
  0.06
 0.05
 0.05
 0.05
 0.04
 0.04
 0.04
 0.03
 0.03
 0.03
 0.03
 0.03
 0.02
 0.02
 0.02
 0.01
 0.01
 0.01
 0.00
 0.00
 0.00
 0.00
 -0.01
 -0.01
 -0.01
 -0.01
 -0.01
  94  Proteins eluted with SDS sample buffer (strong parkin interactors – CCCP conditions) SFRS2B Isoform 1 of Splicing factor, arginine/serine-rich 2B
 HSPA1B;HSPA1A Heat shock 70 kDa protein 1
 NEFM Neurofilament medium polypeptide
 FBXO21 Isoform 1 of F-box only protein 21
 LARS Leucyl-tRNA synthetase, cytoplasmic
 SFPQ Isoform Long of Splicing factor, proline- and glutamine-rich
 HSPA9 Stress-70 protein, mitochondrial
 EEF1AL3;EEF1A1 Putative elongation factor 1-alpha-like 3
 SNRPD2 Small nuclear ribonucleoprotein Sm D2
 RPS4X 40S ribosomal protein S4, X isoform
 EPRS Bifunctional aminoacyl-tRNA synthetase
 PDHA1 Mitochondrial PDHA1
 PSMD13 proteasome 26S non-ATPase subunit 13 isoform 2
 DDX5 Probable ATP-dependent RNA helicase DDX5
 HIST1H1B Histone H1.5
 QPCT Glutaminyl-peptide cyclotransferase
 CCT6A T-complex protein 1 subunit zeta
 TKT 37 kDa protein
 TMOD1 Tropomodulin-1
 DDX3X ATP-dependent RNA helicase DDX3X
 SNRPD3 Small nuclear ribonucleoprotein Sm D3
 FUS Isoform Short of RNA-binding protein FUS
 AHCY Adenosylhomocysteinase
 RBMX cDNA FLJ38696 fis, clone KIDNE2001931, highly similar to HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN G
 FASN Fatty acid synthase
 IMPDH1 inosine monophosphate dehydrogenase 1 isoform a
 NUDC Nuclear migration protein nudC
 RPL23 60S ribosomal protein L23
 DLAT Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial
 NUDT13 Isoform 2 of Nucleoside diphosphate-linked moiety X motif 13
 EMD Emerin
 MDH2 Malate dehydrogenase, mitochondrial
 PIM2 Serine/threonine-protein kinase Pim-2
 NPM1 Isoform 2 of Nucleophosmin
 PABPC1 Isoform 1 of Polyadenylate-binding protein 1
 GMPS GMP synthase [glutamine-hydrolyzing]
 SLC25A11 Mitochondrial 2-oxoglutarate/malate carrier protein
 MAP3K7IP2 Isoform 1 of Mitogen-activated protein kinase kinase kinase 7-interacting protein 2
 SYNCRIP Isoform 1 of Heterogeneous nuclear ribonucleoprotein Q
 PSMD3 26S proteasome non-ATPase regulatory subunit 3
 NCL Isoform 2 of Nucleolin
 VDAC2 Voltage-dependent anion-selective channel protein 2
 YWHAB Isoform Long of 14-3-3 protein beta/alpha
 ZYX Zyxin
 PPIA Peptidyl-prolyl cis-trans isomerase A
 PRPF31 Isoform 1 of U4/U6 small nuclear ribonucleoprotein Prp31
 PRDX1 Peroxiredoxin-1
 GAPDH Glyceraldehyde-3-phosphate dehydrogenase
 PSPC1 Isoform 2 of Paraspeckle component 1
 RUVBL1 Isoform 1 of RuvB-like 1
 RPL24 60S ribosomal protein L24
 HADHA Trifunctional enzyme subunit alpha, mitochondrial
 MYOM2 Myomesin-2
 SPIN3 Isoform 1 of Spindlin-3
 EIF5A Isoform 2 of Eukaryotic translation initiation factor 5A-1
 PHGDH D-3-phosphoglycerate dehydrogenase
 PRPH Isoform 1 of Peripherin
 VDAC3 Isoform 1 of Voltage-dependent anion-selective channel protein 3
 SF3A1 Splicing factor 3 subunit 1
 YWHAZ 14-3-3 protein zeta/delta
 ROD1 ROD1 regulator of differentiation 1
 RALY RNA binding protein, autoantigenic
 ALDOA Fructose-bisphosphate aldolase A
 ARID1B Novel protein (Fragment)
 CCT3 chaperonin containing TCP1, subunit 3 isoform b
 SF3B4 Splicing factor 3B subunit 4
 LDHB L-lactate dehydrogenase B chain
 XRCC5 ATP-dependent DNA helicase 2 subunit 2
 RAN GTP-binding nuclear protein Ran
 NUFIP2 Nuclear fragile X mental retardation-interacting protein 2
 HNRNPH1 Heterogeneous nuclear ribonucleoprotein H
 CPSF6 Isoform 1 of Cleavage and polyadenylation specificity factor subunit 6
 TUBB3 Tubulin beta-3 chain
 PABPC4 Isoform 1 of Polyadenylate-binding protein 4
 TUBB Tubulin beta chain
 C7orf24 C7orf24 protein
 KTN1 Isoform 2 of Kinectin
 SSBP1 Single-stranded DNA-binding protein, mitochondrial
 TRAP1 57 kDa protein
  Protein Mascot Score  # of peptides  log2(heavy /light)  551
 33
 169
 257
 138
 48
 28
 305
 228
 117
 3759
 2084
 1160
 102
 41
 297
 31
 26
 66
 47
 186
 456
 122
  1
 1
 6
 4
 3
 73
 27
 25
 3
 1
 7
 1
 1
 1
 1
 5
 7
 2
 2
 1
 2
 4
 3
  -0.02
 -0.02
 -0.03
 -0.03
 -0.03
 -0.04
 -0.04
 -0.04
 -0.04
 -0.04
 -0.04
 -0.05
 -0.05
 -0.05
 -0.05
 -0.06
 -0.06
 -0.06
 -0.06
 -0.06
 -0.07
 -0.07
 -0.07
  73
 39
 62
 170
 153
  3
 1
 2
 2
 1
  -0.07
 -0.07
 -0.07
 -0.08
 -0.08
  168
 62
 97
 96
 40
 361
 29
 30
 94
 28
 999
 928
 31
 76
 153
 184
 34
 634
 46
 81
 1296
 1679
 105
 1824
 696
 374
 462
 62
 29
 26
 56
 64
 129
 740
 51
 56
 419
 93
 39
 478
 28
 38
 245
 393
 239
 158
 26
 660
 635
 585
 141
  7
 1
 1
 1
 1
 16
 16
 1
 1
 3
 3
 1
 14
 1
 2
 22
 35
 2
 39
 11
 6
 12
 1
 1
 1
 1
 2
 4
 16
 1
 2
 6
 2
 1
 12
 1
 1
 3
 6
 5
 4
 1
 10
 9
 7
 4
 87
 1
 2
 3
 1
  -0.09
 -0.09
 -0.09
 -0.09
 -0.09
 -0.10
 -0.10
 -0.10
 -0.11
 -0.11
 -0.12
 -0.12
 -0.12
 -0.12
 -0.12
 -0.13
 -0.13
 -0.13
 -0.13
 -0.13
 -0.13
 -0.13
 -0.13
 -0.15
 -0.15
 -0.15
 -0.15
 -0.15
 -0.15
 -0.15
 -0.15
 -0.15
 -0.16
 -0.16
 -0.16
 -0.16
 -0.17
 -0.17
 -0.17
 -0.17
 -0.17
 -0.18
 -0.18
 -0.19
 -0.19
 -0.19
 -0.19
 -0.20
 -0.20
 -0.20
 -0.20
  95  Proteins eluted with SDS sample buffer (strong parkin interactors – CCCP conditions) FKBP4 FK506-binding protein 4
 HNRNPU Isoform Short of Heterogeneous nuclear ribonucleoprotein U
 KIAA0562 Protein
 SKP1 Isoform 1 of S-phase kinase-associated protein 1
 SPTBN1 Isoform Long of Spectrin beta chain, brain 1
 RPL26L1 60S ribosomal protein L26-like 1
 CLNS1A Methylosome subunit pICln
 GSTP1 Glutathione S-transferase P
 RPL30 60S ribosomal protein L30
 CCBL2 kynurenine aminotransferase III isoform 3
 HIST1H2BL Histone H2B type 1-L
 PRDX2 Peroxiredoxin-2
 PRDX6 Peroxiredoxin-6
 FLNA filamin A, alpha isoform 1
 EXOSC6 Exosome complex exonuclease MTR3
 MAP3K7IP1 Mitogen-activated protein kinase kinase kinase 7-interacting protein 1
 HPRT1 Hypoxanthine-guanine phosphoribosyltransferase
 XRCC6 ATP-dependent DNA helicase 2 subunit 1
 RPN1 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 67 kDa subunit precursor
 MTHFD1 C-1-tetrahydrofolate synthase, cytoplasmic
 RBM10 Putative uncharacterized protein DKFZp686E2459
 PRPS1L1 Ribose-phosphate pyrophosphokinase 3
 NAP1L1 Nucleosome assembly protein 1-like 1
 HIST2H3D;HIST2H3C;HIST2H3A Histone H3.2
 TUBA1A Tubulin alpha-1A chain
 IARS IARS protein
 SF3B14 Pre-mRNA branch site protein p14
 HIST1H2AE;HIST1H2AB Histone H2A type 1-B
 EEF2 Elongation factor 2
 RBBP4 Histone-binding protein RBBP4
 ARCN1 Putative uncharacterized protein DKFZp686M09245
 SFRS3 Splicing factor, arginine/serine-rich 3
 CDK3 Cell division protein kinase 3
 RARS Isoform Complexed of Arginyl-tRNA synthetase, cytoplasmic
 BAT2 Isoform 1 of Large proline-rich protein BAT2
 CKB Creatine kinase B-type
 NONO Non-POU domain-containing octamer-binding protein
 TAGLN2 Transgelin-2
 LMNA Progerin
 HNRNPAB Isoform 4 of Heterogeneous nuclear ribonucleoprotein A/B
 HSPA6 Heat shock 70 kDa protein 6
 UCHL1 Uncharacterized protein UCHL1
 TCEB2 Transcription elongation factor B polypeptide 2
 DDX1 ATP-dependent RNA helicase DDX1
 NCBP1 Nuclear cap-binding protein subunit 1
 EPHB6 Ephrin type-B receptor 6
 RPLP2 60S acidic ribosomal protein P2
 IMPDH2 Inosine-5'-monophosphate dehydrogenase 2
 HSPE1 10 kDa heat shock protein, mitochondrial
 ENO1 Isoform alpha-enolase of Alpha-enolase
 HNRNPL heterogeneous nuclear ribonucleoprotein L isoform a
 CLIC1 Chloride intracellular channel protein 1
 PRMT1 HMT1 hnRNP methyltransferase-like 2 isoform 1
 PEBP1 Phosphatidylethanolamine-binding protein 1
 RUVBL2 RuvB-like 2
 CTSB Cathepsin B
 PPP1CA protein phosphatase 1, catalytic subunit, alpha isoform 2
 SET Isoform 1 of Protein SET
 SPIN1 Spindlin-1
 RIOK1 Serine/threonine-protein kinase RIO1
 HNRNPA2B1 Isoform B1 of Heterogeneous nuclear ribonucleoproteins A2/B1
 MAP3K7 Isoform 1A of Mitogen-activated protein kinase kinase kinase 7
 ACTN1 Alpha-actinin-1
 ACTL6A Isoform 1 of Actin-like protein 6A
 HADHB Trifunctional enzyme subunit beta, mitochondrial
 DNAJB11 DnaJ homolog subfamily B member 11
 PSMA4 20 kDa protein
 HSD17B10 Isoform 1 of 3-hydroxyacyl-CoA dehydrogenase type-2
 U2AF2 Splicing factor U2AF 65 kDa subunit
 ASS1 Argininosuccinate synthase
 TUBB2A Tubulin beta-2A chain
 UQCRC1 Cytochrome b-c1 complex subunit 1, mitochondrial
 RPL21;LOC729402 60S ribosomal protein L21
 RCN2 Reticulocalbin-2
 IGF2BP3 Isoform 2 of Insulin-like growth factor 2 mRNA-binding protein 3
 RBBP7 Histone-binding protein RBBP7
 WARS Tryptophanyl-tRNA synthetase, cytoplasmic
 LOC144097 Uncharacterized protein LOC144097
 U2AF1L4 Isoform 2 of Splicing factor U2AF 26 kDa subunit
 SFRS1 Isoform ASF-1 of Splicing factor, arginine/serine-rich 1
 SDF2L1 Dihydropyrimidinase-like 2
  Protein Mascot Score  # of peptides  log2(heavy /light)  5058
 32
 73
 98
 36
 62
 1021
 39
 95
 173
 33
 350
 747
 199
 26
 821
 246
 629
 3077
 327
 2478
 38
 29
 43
 58
 1055
 31
 66
 210
 2843
 36
 25
 28
 218
 42
 213
 99
 63
 118
 31
 308
 9113
 29
 1208
 69
 33
 155
 147
 59
 40
 29
 44
 297
 107
 719
 209
 35
 165
 118
 401
 32
 45
 310
 1233
 433
 305
 648
 28
 26
 84
 73
 35
 39
 84
 32
 27
 119
 53
 30
 26
 39
  2
 14
 1
 2
 5
 1
 7
 9
 3
 1
 12
 6
 11
 57
 7
 44
 1
 1
 1
 1
 22
 1
 2
 7
 50
 1
 1
 1
 6
 1
 4
 2
 1
 3
 1
 6
 161
 1
 23
 1
 1
 3
 3
 2
 1
 1
 1
 7
 2
 14
 5
 1
 4
 3
 7
 1
 1
 4
 23
 8
 5
 11
 1
 1
 2
 2
 1
 1
 2
 1
 1
 2
 1
 1
 1
 1
 1
 8
 2
 2
 1
  -0.21
 -0.22
 -0.22
 -0.22
 -0.22
 -0.22
 -0.22
 -0.22
 -0.23
 -0.24
 -0.24
 -0.24
 -0.25
 -0.25
 -0.25
 -0.26
 -0.26
 -0.26
 -0.26
 -0.26
 -0.27
 -0.27
 -0.27
 -0.27
 -0.29
 -0.30
 -0.30
 -0.32
 -0.33
 -0.33
 -0.33
 -0.33
 -0.33
 -0.33
 -0.33
 -0.34
 -0.34
 -0.34
 -0.34
 -0.34
 -0.36
 -0.36
 -0.36
 -0.37
 -0.38
 -0.39
 -0.41
 -0.41
 -0.41
 -0.42
 -0.43
 -0.43
 -0.45
 -0.46
 -0.47
 -0.47
 -0.48
 -0.48
 -0.49
 -0.50
 -0.50
 -0.50
 -0.51
 -0.51
 -0.51
 -0.51
 -0.52
 -0.53
 -0.54
 -0.55
 -0.57
 -0.58
 -0.60
 -0.63
 -0.63
 -0.63
 -0.68
 -0.69
 -0.69
 -0.69
 -0.70
  96  Proteins eluted with SDS sample buffer (strong parkin interactors – CCCP conditions) CDY1;CDY1B Isoform 1 of Testis-specific chromodomain protein Y 1
 HNRNPM Isoform 1 of Heterogeneous nuclear ribonucleoprotein M
 PFN1 Profilin-1
 STRAP Serine-threonine kinase receptor-associated protein
 PGK1 Phosphoglycerate kinase 1
 OTUD4 Isoform 1 of OTU domain-containing protein 4
 DHX9 ATP-dependent RNA helicase A
 TAF4 Transcription initiation factor TFIID subunit 4
 PDHB Isoform 1 of Pyruvate dehydrogenase E1 component subunit beta, mitochondrial
 LUC7L Isoform 1 of Putative RNA-binding protein Luc7-like 1
 SEPT9 Isoform 3 of Septin-9
 KIF11 Kinesin-like protein KIF11
 LMNB1 Lamin-B1
 CDK5RAP1 Isoform 4 of CDK5 regulatory subunit-associated protein 1
 BOLA2;BOLA2B BolA-like protein 2
 HNRNPC Isoform C1 of Heterogeneous nuclear ribonucleoproteins C1/C2
 LOC729708;LOC388642 Triosephosphate isomerase (Fragment)
 HDAC2 histone deacetylase 2
 STK38L Serine/threonine-protein kinase 38-like
 STK38 Serine/threonine-protein kinase 38
 CRKL Crk-like protein
 SMC4 Isoform 2 of Structural maintenance of chromosomes protein 4
 ERH Enhancer of rudimentary homolog
 EIF6 Eukaryotic translation initiation factor 6
 USP29 Ubiquitin carboxyl-terminal hydrolase 29
 PCNA Proliferating cell nuclear antigen
 MTHFD1L Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like
 TSC22D4 TSC22 domain family protein 4
 COX15 Isoform 1 of Cytochrome c oxidase assembly protein COX15 homolog
 CTNNB1 Isoform 1 of Catenin beta-1
 SF3B1 Splicing factor 3B subunit 1
 ARHGEF11 Rho guanine nucleotide exchange factor 11
 ACTR2 Actin-related protein 2
 RNF7 Isoform 1 of RING-box protein 2
 A26C1B ANKRD26-like family C member 1B
 NUMA1 Isoform 2 of Nuclear mitotic apparatus protein 1
 SLFN13 Isoform 1 of Schlafen family member 13
 DHRSX 29 kDa protein
 LOC550643 Putative uncharacterized protein LOC550643
 GOLGB1 Golgin subfamily B member 1
 FUT3 Galactoside 3(4)-L-fucosyltransferase
 CCT4 T-complex protein 1 subunit delta
 C1orf65 Uncharacterized protein C1orf65
 SNRPB Isoform SM-B' of Small nuclear ribonucleoprotein-associated proteins B and B'
 ZNF681 Zinc finger protein 681
 PARK2 Isoform 1 of E3 ubiquitin-protein ligase parkin
 LOC646730 similar to hCG2036631
 CDADC1 Cytidine and dCMP deaminase domain containing 1
 LOC388720 similar to ubiquitin
 - Uncharacterized protein ENSP00000379605
 Uncharacterized protein ENSP00000381458 (Fragment)
 TCFL5 Isoform 1 of Transcription factor-like 5 protein
 METTL8 methyltransferase like 8
 HPX-2 similar to facioscapulohumeral muscular dystrophy
 PCM1 Isoform 3 of Pericentriolar material 1 protein
 LOC100130931 cDNA FLJ46693 fis, clone TRACH3012864
 PDZD3 Isoform 2 of PDZ domain-containing protein 3
  Protein Mascot Score  # of peptides  log2(heavy /light)  94
 367
 82
 71
 67
 29
 582
 223
 55
 59
 114
 70
 47
 35
 135
 50
 4027
 267
 25
 69
 771
 105
 69
 177
 431
 64
 59
 147
 252
 56
 67
 28
 25
 26
 25
 117
 26
 25
 35
 37
 59
 28
 28
 25
 57
 25
 33
 27
 26
 110
 228
 40
 56
 33
 25
 60
 28
  1
 10
 4
 1
 2
 3
 2
 1
 1
 2
 1
 70
 5
 1
 1
 11
 2
 2
 5
 9
 1
 2
 3
 5
 2
 1
 1
 1
 1
 1
 2
 1
 1
 1
 1
 2
 1
 1
 1
 2
 1
 1
 1
 1
 4
 4
 1
 1
 1
 1
 2
 1
 1
 1
 6
 1
 1
  -0.70
 -0.71
 -0.72
 -0.72
 -0.75
 -0.76
 -0.83
 -0.83
 -0.83
 -0.84
 -0.85
 -0.86
 -0.89
 -0.89
 -0.89
 -0.92
 -0.93
 -0.93
 -0.95
 -0.97
 -1.00
 -1.01
 -1.01
 -1.03
 -1.05
 -1.12
 -1.26
 -1.35
 -1.40
 -1.41
 -1.43
 -1.45
 -1.46
 -1.64
 -1.74
 -1.77
 -1.79
 -1.86
 -2.16
 -2.21
 -2.22
 -2.29
 -2.50
 -2.54
 -2.58
 -2.74
 -2.78
 -2.81
 -3.00
 -3.06
 -3.13
 -3.13
 -3.54
 -3.82
 -4.27
 -4.38
 -4.50
  97  Table 5. FLAG-parkin pull-down proteins under CCCP with SILAC ratios – weak interactor fraction Proteins eluted with mild detergent and salt (weak parkin interactors – CCCP conditions) DNAJC28 J domain-containing protein C21orf55
 WDR82 WD repeat-containing protein 82
 LOC654340 similar to KIAA1839 protein
 VDAC3 Isoform 2 of Voltage-dependent anion-selective channel protein 3
 TOP1 DNA topoisomerase 1
 HNRNPM Isoform 1 of Heterogeneous nuclear ribonucleoprotein M
 MCCC2 Isoform 1 of Methylcrotonoyl-CoA carboxylase beta chain, mitochondrial
 FKBP1A Peptidyl-prolyl cis-trans isomerase
 MAN2B2 Isoform 1 of Epididymis-specific alpha-mannosidase
 STK38 Serine/threonine-protein kinase 38
 KCTD5 BTB/POZ domain-containing protein KCTD5
 SDF4 stromal cell derived factor 4 precursor
 SYCP1 Synaptonemal complex protein 1
 PPM1B Isoform Beta-1 of Protein phosphatase 1B
 ITGBL1 Integrin beta-like protein 1
 STK32C Protein kinase
 DHX38 Pre-mRNA-splicing factor ATP-dependent RNA helicase PRP16
 DDX50 ATP-dependent RNA helicase DDX50
 CASK Isoform 3 of Peripheral plasma membrane protein CASK
 PKN3 Serine/threonine-protein kinase N3
 DDX60L DEAD (Asp-Glu-Ala-Asp) box polypeptide 60-like
 OSGEP Probable O-sialoglycoprotein endopeptidase
 LOC729176 LOC729176 protein (Fragment)
 RPL32 60S ribosomal protein L32
 CBFA2T2 Isoform 1 of Protein CBFA2T2
 ITPR3 Inositol 1,4,5-trisphosphate receptor type 3
 LOC203510 similar to hCG1644442
 ASCC3L1 Isoform 1 of U5 small nuclear ribonucleoprotein 200 kDa helicase
 C21orf114 Putative transposase element C21orf114
 DDB1 DNA damage-binding protein 1
 FBXL21 F-box/LRR-repeat protein 21
 LAMB1 Laminin subunit beta-1
 PARP1 Poly [ADP-ribose] polymerase 1
 PSMA1 Isoform Short of Proteasome subunit alpha type-1
 PSMA2 Proteasome subunit alpha type-2
 TMED9 transmembrane emp24 protein transport domain containing 9
 DCI Isoform 1 of 3,2-trans-enoyl-CoA isomerase, mitochondrial
 EIF3D Eukaryotic translation initiation factor 3 subunit D
 CIRBP Cold-inducible RNA-binding protein
 MYH9 Myosin-9
 WDR77 Methylosome protein 50
 PPME1 Isoform 1 of Protein phosphatase methylesterase 1
 SMC3 Structural maintenance of chromosomes protein 3
 OSBPL8 Oxysterol-binding protein
 ATP5D ATP synthase subunit delta, mitochondrial
 PSMA7 Isoform 1 of Proteasome subunit alpha type-7
 HP1BP3 Isoform 5 of Heterochromatin protein 1-binding protein 3
 NARS Asparaginyl-tRNA synthetase, cytoplasmic
 PRIC285 Isoform 1 of Peroxisomal proliferator-activated receptor A-interacting complex 285 kDa 
 EIF3A Eukaryotic translation initiation factor 3 subunit A
 BCCIP Isoform 1 of BRCA2 and CDKN1A-interacting protein
 SDHB Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial
 SART3 Isoform 1 of Squamous cell carcinoma antigen recognized by T-cells 3
 FTH1 Ferritin heavy chain
 EIF3EIP Eukaryotic translation initiation factor 3, subunit E interacting protein
 DYNC1H1 Cytoplasmic dynein 1 heavy chain 1
 EIF4B Eukaryotic translation initiation factor 4B
 HNRNPR heterogeneous nuclear ribonucleoprotein R isoform 3
 CHGB Secretogranin-1
 ICT1 Immature colon carcinoma transcript 1 protein
 EIF3G Eukaryotic translation initiation factor 3 subunit G
 TMOD3 Tropomodulin-3
 LMNB1 Lamin-B1
 SPIN1 Spindlin-1
 MAP3K7IP1 Mitogen-activated protein kinase kinase kinase 7-interacting protein 1
 PSMA5 Proteasome subunit alpha type-5
 THOC3 THO complex subunit 3
 DPYSL5 Dihydropyrimidinase-related protein 5
 YARS2 Tyrosyl-tRNA synthetase, mitochondrial
 UBE2O Ubiquitin-conjugating enzyme E2 O
 LSM14A Isoform 2 of LSM14 protein homolog A
 RP11-631M21.2 Tubulin beta-8 chain
 RUVBL2 RuvB-like 2
 NUP153 Nuclear pore complex protein Nup153
 RBM25 RNA binding motif protein 25
 HDGF2 Isoform 2 of Hepatoma-derived growth factor-related protein 2
  Protein Mascot Score  # of peptides  log2(heavy /light)  52
 65
 57
 190
 71
 208
 90
 205
 54
 31
 45
 64
 27
 86
 25
 30
 34
 39
 32
 29
 26
 45
 32
 168
 27
 27
 32
 121
 58
 56
 34
 106
 459
 114
 189
 86
 58
 254
 26
 967
 170
 26
 29
 40
 94
 118
 64
 155
 25
 542
 47
 133
 42
 226
 134
 574
 439
 120
 475
 57
 46
 107
 32
 37
 325
 118
 48
 401
 32
 67
 62
 31
 233
 31
 26
 118
  2
 2
 2
 4
 2
 5
 2
 3
 2
 1
 1
 1
 1
 2
 1
 1
 1
 1
 1
 1
 1
 1
 1
 3
 1
 1
 1
 4
 2
 1
 1
 3
 7
 3
 3
 1
 1
 5
 1
 18
 2
 1
 1
 1
 1
 2
 1
 3
 1
 14
 1
 4
 1
 5
 2
 11
 9
 3
 8
 1
 1
 2
 1
 1
 7
 3
 1
 9
 1
 1
 1
 1
 4
 1
 1
 2
  3.14
 1.89
 1.59
 1.46
 1.27
 1.15
 1.12
 1.07
 0.98
 0.98
 0.98
 0.97
 0.95
 0.94
 0.91
 0.91
 0.85
 0.85
 0.85
 0.84
 0.84
 0.82
 0.82
 0.80
 0.79
 0.77
 0.74
 0.73
 0.72
 0.72
 0.71
 0.70
 0.63
 0.61
 0.61
 0.60
 0.55
 0.54
 0.52
 0.52
 0.51
 0.51
 0.51
 0.50
 0.50
 0.50
 0.48
 0.48
 0.48
 0.47
 0.47
 0.47
 0.47
 0.46
 0.45
 0.44
 0.44
 0.44
 0.44
 0.43
 0.42
 0.42
 0.41
 0.40
 0.39
 0.39
 0.38
 0.38
 0.37
 0.36
 0.36
 0.35
 0.35
 0.34
 0.34
 0.33
  98  Proteins eluted with mild detergent and salt (weak parkin interactors – CCCP conditions) TTYH3 Isoform 1 of Protein tweety homolog 3
 HGS Hepatocyte growth factor-regulated tyrosine kinase substrate
 TLN1 Talin-1
 LAMA1 Laminin subunit alpha-1
 PDCD4 programmed cell death 4 isoform 2
 ABLIM1 Isoform 1 of Actin-binding LIM protein 1
 GALNT2 Polypeptide N-acetylgalactosaminyltransferase 2
 MARCKSL1 MARCKS-related protein
 SEC22B Vesicle-trafficking protein SEC22b
 POP1 Ribonucleases P/MRP protein subunit POP1
 GNRHR Isoform 1 of Gonadotropin-releasing hormone receptor
 IMMT Isoform 1 of Mitochondrial inner membrane protein
 MRLC2 Myosin regulatory light chain
 ACTN1 Alpha-actinin-1
 PTGIS Prostacyclin synthase
 CKAP4 Isoform 1 of Cytoskeleton-associated protein 4
 MSI2 Isoform 1 of RNA-binding protein Musashi homolog 2
 RPS13 40S ribosomal protein S13
 RPS26 Ribosomal protein 26 (RPS26) pseudogene
 CKMT1B;CKMT1A;LOC100133623 Creatine kinase, ubiquitous mitochondrial
 PRMT5 Protein arginine N-methyltransferase 5
 PSMC3 26S protease regulatory subunit 6A
 EIF3J Eukaryotic translation initiation factor 3 subunit J
 RPL28 60S ribosomal protein L28
 EIF2S1 Eukaryotic translation initiation factor 2 subunit 1
 CNPY2 Isoform 2 of Protein canopy homolog 2
 PSMD11 Proteasome 26S non-ATPase subunit 11 variant (Fragment)
 HGSNAT Heparan-alpha-glucosaminide N-acetyltransferase
 RPS18;LOC100130553 40S ribosomal protein S18
 RCC2 Protein RCC2
 ARPC1B Actin-related protein 2/3 complex subunit 1B
 NUCKS1 Isoform 1 of Nuclear ubiquitous casein and cyclin-dependent kinases substrate
 H1FX Histone H1x
 RBM10 Putative uncharacterized protein DKFZp686E2459
 ATP2A2 Isoform SERCA2A of Sarcoplasmic/endoplasmic reticulum calcium ATPase 2
 XRCC5 ATP-dependent DNA helicase 2 subunit 2
 HIST1H1C Histone H1.2
 BAX Isoform Zeta of Apoptosis regulator BAX
 CTSD 20 kDa protein
 EIF3F Eukaryotic translation initiation factor 3 subunit 5
 CYB5R3 Isoform 1 of NADH-cytochrome b5 reductase 3
 RPL31 60S ribosomal protein L31
 NDUFV2 NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial
 HMGA1 Isoform HMG-Y of High mobility group protein HMG-I/HMG-Y
 TOR1AIP1 Isoform 1 of Torsin-1A-interacting protein 1
 XRCC6 ATP-dependent DNA helicase 2 subunit 1
 HDAC1 Histone deacetylase 1
 EIF4A2 BM-010
 PDAP1 28 kDa heat- and acid-stable phosphoprotein
 RPS10 40S ribosomal protein S10
 EIF2S2 Eukaryotic translation initiation factor 2 subunit 2
 CALD1 Isoform 1 of Caldesmon
 MYL6;MYL6B Isoform Non-muscle of Myosin light polypeptide 6
 ILF3 Isoform 5 of Interleukin enhancer-binding factor 3
 CCT6A T-complex protein 1 subunit zeta
 RPS17 40S ribosomal protein S17
 MAP1LC3A Isoform 2 of Microtubule-associated proteins 1A/1B light chain 3A
 NDUFV1 Isoform 1 of NADH dehydrogenase [ubiquinone] flavoprotein 1, mitochondrial
 PFKM Isoform 2 of 6-phosphofructokinase, muscle type
 EIF6 Eukaryotic translation initiation factor 6
 - 46 kDa protein
 CABC1 Isoform 1 of Chaperone activity of bc1 complex-like, mitochondrial
 HSPD1 60 kDa heat shock protein, mitochondrial
 HSPD1 60 kDa heat shock protein, mitochondrial
 IQGAP3 Ras GTPase-activating-like protein IQGAP3
 RPS19 40S ribosomal protein S19
 SATB1 DNA-binding protein SATB1
 SLC25A6 ADP/ATP translocase 3
 MYH10 Isoform 1 of Myosin-10
 H2AFV Histone H2A.V
 TIMM13 Mitochondrial import inner membrane translocase subunit Tim13
 RPS15 40S ribosomal protein S15
 ADNP Activity-dependent neuroprotector homeobox protein
 HSPA5 HSPA5 protein
 HSPA5 HSPA5 protein
 RPS3 40S ribosomal protein S3
 ACTN4 Alpha-actinin-4
 ENSA Isoform 2 of Alpha-endosulfine
 STXBP1 Isoform 2 of Syntaxin-binding protein 1
 RPS4X 40S ribosomal protein S4, X isoform
 PSPC1 Isoform 2 of Paraspeckle component 1
  Protein Mascot Score  # of peptides  log2(heavy /light)  70
 39
 110
 28
 85
 250
 80
 97
 36
 27
 26
 84
 210
 561
 25
 288
 87
 83
 203
 423
 1270
 397
 250
 77
 252
 26
 30
 28
 330
 156
 38
 298
 186
 35
 210
 1542
 524
 31
 44
 658
 241
 195
 126
 66
 29
 34
 30
 29
 132
 490
 69
 70
 187
 623
 550
 292
 39
 86
 486
 60
 54
 26
 6486
 6486
 31
 435
 37
 492
 514
 54
 156
 849
 29
 3297
 3297
 1476
 1355
 50
 75
 1244
 281
  1
 1
 3
 1
 3
 5
 2
 2
 1
 1
 1
 3
 5
 12
 1
 4
 2
 1
 6
 9
 27
 6
 3
 2
 6
 1
 1
 1
 8
 4
 1
 3
 3
 1
 4
 25
 10
 1
 1
 9
 5
 4
 2
 1
 1
 1
 1
 1
 2
 8
 2
 2
 5
 13
 12
 6
 1
 3
 11
 1
 2
 1
 120
 120
 1
 10
 1
 11
 10
 1
 2
 13
 1
 56
 56
 32
 29
 1
 1
 24
 7
  0.33
 0.33
 0.32
 0.32
 0.32
 0.31
 0.30
 0.30
 0.30
 0.29
 0.29
 0.28
 0.28
 0.28
 0.28
 0.27
 0.26
 0.26
 0.25
 0.25
 0.25
 0.25
 0.25
 0.25
 0.24
 0.24
 0.23
 0.23
 0.22
 0.22
 0.21
 0.20
 0.20
 0.20
 0.19
 0.19
 0.19
 0.18
 0.18
 0.18
 0.18
 0.18
 0.17
 0.17
 0.17
 0.17
 0.17
 0.16
 0.16
 0.15
 0.15
 0.14
 0.14
 0.12
 0.12
 0.12
 0.12
 0.12
 0.11
 0.11
 0.11
 0.10
 0.10
 0.10
 0.10
 0.10
 0.09
 0.09
 0.08
 0.08
 0.08
 0.08
 0.08
 0.08
 0.08
 0.08
 0.08
 0.08
 0.08
 0.07
 0.07
  99  Proteins eluted with mild detergent and salt (weak parkin interactors – CCCP conditions) BBS1;DPP3 Isoform 1 of Dipeptidyl-peptidase 3
 EIF3CL;EIF3C Eukaryotic translation initiation factor 3 subunit C
 LOC220429 Similar to CTAGE family, member 5 isoform 2
 QDPR Dihydropteridine reductase
 UQCRC1 Cytochrome b-c1 complex subunit 1, mitochondrial
 GLG1 golgi apparatus protein 1
 AP1B1 Isoform A of AP-1 complex subunit beta-1
 TUBAL3 Isoform 1 of Tubulin alpha chain-like 3
 IARS Isoleucyl-tRNA synthetase, cytoplasmic
 DNMT1 Isoform 1 of DNA (cytosine-5)-methyltransferase 1
 KTN1 Isoform 1 of Kinectin
 ATP5A1 ATP synthase subunit alpha, mitochondrial
 PRDX2 Peroxiredoxin-2
 PGRMC1 Membrane-associated progesterone receptor component 1
 SNRPG Small nuclear ribonucleoprotein G
 SEPT2 Septin-2
 RPL22 60S ribosomal protein L22
 HEATR2 Isoform 1 of HEAT repeat-containing protein 2
 RPS14 40S ribosomal protein S14
 GNB2L1 Lung cancer oncogene 7
 APEX1 DNA-(apurinic or apyrimidinic site) lyase
 DPP7 Dipeptidyl-peptidase 2
 RPS20 40S ribosomal protein S20
 EDF1 Isoform 2 of Endothelial differentiation-related factor 1
 PSMD2 26S proteasome non-ATPase regulatory subunit 2
 RPS23 40S ribosomal protein S23
 AK2 Isoform 1 of Adenylate kinase isoenzyme 2, mitochondrial
 HIST1H2BD Histone H2B type 1-D
 CORO1C Coronin-1C_i3 protein
 BZW2 Basic leucine zipper and W2 domain-containing protein 2
 MPST 3-mercaptopyruvate sulfurtransferase
 PRKAR2A PRKAR2A protein
 MYBL2 Uncharacterized protein MYBL2 (Fragment)
 MYO1B Isoform 1 of Myosin-Ib
 FARSA Phenylalanyl-tRNA synthetase alpha chain
 HIST2H4B;HIST1H4F;HIST1H4J;HIST1H4C;HIST1H4B;HIST1H4K;HIST1H4E;HIST2H4A;HIST1 H4H;HIST1H4L;HIST1H4A;HIST4H4;HIST1H4D;HIST1H4I Histone H4
 ERO1L ERO1-like protein alpha
 SEPT11 Septin-11
 CCT5 T-complex protein 1 subunit epsilon
 RPS16 40S ribosomal protein S16
 DLST Full-length cDNA 5-PRIME end of clone CS0DB006YE12 of Neuroblastoma of H. sapiens
 THOC4 THO complex subunit 4
 RPS9 40S ribosomal protein S9
 EIF5B Eukaryotic translation initiation factor 5B
 CCDC102B Isoform 1 of Coiled-coil domain-containing protein 102B
 LENG8 Isoform 1 of Leukocyte receptor cluster member 8
 PC Pyruvate carboxylase, mitochondrial
 BUB3 Mitotic checkpoint protein BUB3
 FASN Fatty acid synthase
 FASN Fatty acid synthase
 - Uncharacterized protein ENSP00000343748
 ACAT1 Acetyl-CoA acetyltransferase, mitochondrial
 PRDX4 Peroxiredoxin-4
 PGD 6-phosphogluconate dehydrogenase, decarboxylating
 PPP1CC Isoform Gamma-1 of Serine/threonine-protein phosphatase PP1-gamma catalytic subunit
 EIF3E Eukaryotic translation initiation factor 3 subunit E
 ACLY ATP-citrate synthase
 DBN1 Isoform 1 of Drebrin
 CTSC Dipeptidyl-peptidase 1
 PDCD6IP PDCD6IP protein
 ATP1A3 Sodium/potassium-transporting ATPase subunit alpha-3
 HSP90B1 Endoplasmin
 TUBB1 Tubulin beta-1 chain
 RUVBL1 Isoform 1 of RuvB-like 1
 SLC25A3 Isoform A of Phosphate carrier protein, mitochondrial
 NME2 Nucleoside diphosphate kinase
 FUBP1 Isoform 1 of Far upstream element-binding protein 1
 TH Tyrosine hydroxylase isoform D2,8,9
 PPP2CA Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform
 DNPEP aspartyl aminopeptidase
 GNS N-acetylglucosamine-6-sulfatase
 UBFD1 Ubiquitin domain-containing protein UBFD1
 HIST1H1B Histone H1.5
 PPAT Amidophosphoribosyltransferase
 MAP2 Isoform 1 of Microtubule-associated protein 2
 PSMB1 Proteasome subunit beta type-1
 ME2 NAD-dependent malic enzyme, mitochondrial
 EIF3B Isoform 1 of Eukaryotic translation initiation factor 3 subunit B
 NUDT5 ADP-sugar pyrophosphatase
 DPYSL3 DPYSL3 protein
  Protein Mascot Score  # of peptides  log2(heavy /light)  38
 499
 28
 158
 160
 65
 63
 91
 132
 61
 32
 1748
 811
 178
 26
 1046
 223
 55
 469
 1138
 347
 320
 145
 31
 831
 370
 29
 770
 202
 29
 242
 49
 64
 146
 574
  1
 10
 1
 3
 3
 2
 2
 1
 2
 1
 1
 30
 18
 4
 1
 21
 5
 2
 9
 18
 7
 7
 3
 1
 16
 8
 1
 14
 3
 1
 4
 1
 2
 4
 10
  0.07
 0.07
 0.07
 0.06
 0.06
 0.06
 0.06
 0.06
 0.06
 0.06
 0.06
 0.05
 0.05
 0.05
 0.05
 0.05
 0.05
 0.05
 0.05
 0.04
 0.04
 0.04
 0.04
 0.04
 0.04
 0.04
 0.04
 0.03
 0.03
 0.03
 0.03
 0.03
 0.03
 0.03
 0.02
  405
 208
 882
 1239
 157
 334
 450
 469
 30
 73
 35
 315
 139
 2790
 2790
 1312
 1971
 1246
 806
 155
 53
 2278
 678
 82
 85
 28
 1958
 143
 277
 187
 1706
 359
 202
 301
 46
 29
 27
 311
 288
 203
 132
 37
 613
 40
 67
  9
 4
 15
 23
 5
 4
 9
 13
 1
 2
 1
 6
 3
 60
 60
 25
 29
 21
 9
 4
 2
 39
 11
 2
 2
 1
 40
 4
 7
 4
 32
 8
 4
 6
 1
 1
 1
 7
 6
 3
 2
 1
 13
 1
 2
  0.02
 0.02
 0.02
 0.02
 0.02
 0.01
 0.01
 0.01
 0.01
 0.01
 0.01
 0.01
 0.01
 0.01
 0.01
 0.01
 0.01
 0.01
 0.01
 0.01
 0.01
 0.00
 0.00
 0.00
 0.00
 0.00
 0.00
 0.00
 0.00
 0.00
 -0.01
 -0.01
 -0.01
 -0.01
 -0.01
 -0.01
 -0.01
 -0.01
 -0.01
 -0.01
 -0.01
 -0.01
 -0.02
 -0.02
 -0.02
  100  Proteins eluted with mild detergent and salt (weak parkin interactors – CCCP conditions) SCG2 Secretogranin-2
 LBR Lamin-B receptor
 XRCC4 Isoform 1 of DNA-repair protein XRCC4
 DDX17 DEAD box polypeptide 17 isoform 1
 CALU Isoform 2 of Calumenin
 RPL36AL 60S ribosomal protein L36a-like
 APP Isoform APP770 of Amyloid beta A4 protein (Fragment)
 RPS11 40S ribosomal protein S11
 RPS15A 40S ribosomal protein S15a
 CBR1 Carbonyl reductase [NADPH] 1
 MTCH2 Mitochondrial carrier homolog 2
 TFRC 6 kDa protein
 CAPZB Isoform 1 of F-actin-capping protein subunit beta
 RPL15 22 kDa protein
 NIPSNAP1 Protein NipSnap homolog 1
 HPRT1 Hypoxanthine-guanine phosphoribosyltransferase
 PSMD4 Isoform Rpn10A of 26S proteasome non-ATPase regulatory subunit 4
 SFPQ Isoform Long of Splicing factor, proline- and glutamine-rich
 LOC442497;SLC3A2 4F2 cell-surface antigen heavy chain
 ATP5B ATP synthase subunit beta, mitochondrial
 ATP5B ATP synthase subunit beta, mitochondrial
 TCP1 T-complex protein 1 subunit alpha
 PDHB Isoform 1 of Pyruvate dehydrogenase E1 component subunit beta, mitochondrial
 DEK Protein DEK
 RPL23A;hCG_16001 60S ribosomal protein L23a
 MAP1B Microtubule-associated protein 1B
 PRPS1 Ribose-phosphate pyrophosphokinase 1
 P4HB Protein disulfide-isomerase
 MTHFD1L Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like
 CPSF6 Isoform 1 of Cleavage and polyadenylation specificity factor subunit 6
 NME1;NME2 non-metastatic cells 1, protein (NM23A) expressed in isoform a
 TCEA1 Isoform 2 of Transcription elongation factor A protein 1
 NPEPPS Puromycin-sensitive aminopeptidase
 TARS Threonyl-tRNA synthetase, cytoplasmic
 SEC23A Protein transport protein Sec23A
 IDH3A Isoform 1 of Isocitrate dehydrogenase [NAD] subunit alpha, mitochondrial
 ALDOA Fructose-bisphosphate aldolase A
 ALDOA Fructose-bisphosphate aldolase A
 TUBB3 Tubulin beta-3 chain
 CFL1 Cofilin-1
 CRIP2 Cysteine-rich protein 2
 UMODL1 Uromodulin-like 1 protein variant 6
 YWHAQ 14-3-3 protein theta
 LMAN2 Vesicular integral-membrane protein VIP36
 PPP2R2A Serine/threonine-protein phosphatase 2A 55 kDa regulatory subunit B alpha isoform
 ATP1B1 Isoform 1 of Sodium/potassium-transporting ATPase subunit beta-1
 ACTG1 Actin, cytoplasmic 2
 ACTG1 Actin, cytoplasmic 2
 NLRC3 Isoform 4 of Protein NLRC3
 GTPBP1 GTP-binding protein 1
 IPO9 Importin-9
 TXNRD1 Isoform 5 of Thioredoxin reductase 1, cytoplasmic
 TOMM40 Isoform 1 of Mitochondrial import receptor subunit TOM40 homolog
 SUB1 Activated RNA polymerase II transcriptional coactivator p15
 NDUFS1 NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial
 USP7 Ubiquitin carboxyl-terminal hydrolase 7
 DDX19B Isoform 1 of ATP-dependent RNA helicase DDX19B
 PHB2 Prohibitin-2
 IQGAP1 Ras GTPase-activating-like protein IQGAP1
 VCP Transitional endoplasmic reticulum ATPase
 RPN1 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 67 kDa subunit precursor
 NAP1L1 Nucleosome assembly protein 1-like 1
 ARCN1 Putative uncharacterized protein DKFZp686M09245
 OGDH 2-oxoglutarate dehydrogenase E1 component, mitochondrial
 MUTED;TXNDC5 Thioredoxin domain-containing protein 5
 SDHA Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial
 PPP1R7 Protein
 FLNA filamin A, alpha isoform 1
 FLNA filamin A, alpha isoform 1
 GARS Glycyl-tRNA synthetase
 RPS24 Isoform 1 of 40S ribosomal protein S24
 XTP3TPA XTP3-transactivated gene A protein
 HN1L Isoform 1 of Hematological and neurological expressed 1-like protein
 SRP54 Signal recognition particle 54 kDa protein
 RPS8 25 kDa protein
 PSMC2 26S protease regulatory subunit 7
 PFDN2 Prefoldin subunit 2
 CENPN Isoform 2 of Centromere protein N
 C19orf63 Isoform 2 of UPF0510 protein C19orf63
 SKIV2L2 Superkiller viralicidic activity 2-like 2
 CANX Calnexin
  Protein Mascot Score  # of peptides  log2(heavy /light)  859
 407
 26
 376
 330
 41
 25
 597
 323
 222
 94
 33
 686
 192
 58
 107
 88
 1041
 189
 4746
 4746
 1533
 65
 95
 170
 2793
 344
 303
 58
 103
 144
 38
 113
 509
 66
 197
 4093
 4093
 1683
 1001
 220
 26
 1032
 211
 211
 47
 7343
 7343
 29
 139
 27
 185
 100
 50
 69
 32
 31
 433
 120
 1783
 892
 406
 192
 118
 144
 788
 62
 5079
 5079
 1175
 578
 293
 94
 47
 1074
 542
 27
 27
 26
 85
 735
  15
 8
 1
 8
 9
 1
 1
 13
 7
 4
 2
 1
 16
 4
 1
 3
 2
 22
 3
 70
 70
 31
 1
 3
 4
 48
 6
 7
 2
 2
 2
 1
 3
 10
 1
 6
 72
 72
 28
 14
 4
 1
 13
 4
 4
 1
 128
 128
 1
 3
 1
 3
 2
 1
 2
 1
 1
 8
 3
 29
 17
 7
 2
 3
 4
 18
 1
 98
 98
 22
 12
 4
 2
 1
 17
 10
 1
 1
 1
 2
 13
  -0.02
 -0.02
 -0.02
 -0.02
 -0.02
 -0.02
 -0.03
 -0.03
 -0.03
 -0.03
 -0.03
 -0.03
 -0.03
 -0.03
 -0.03
 -0.03
 -0.03
 -0.04
 -0.04
 -0.04
 -0.04
 -0.04
 -0.04
 -0.04
 -0.04
 -0.05
 -0.05
 -0.05
 -0.05
 -0.05
 -0.05
 -0.05
 -0.06
 -0.06
 -0.06
 -0.06
 -0.06
 -0.06
 -0.06
 -0.06
 -0.06
 -0.06
 -0.06
 -0.06
 -0.07
 -0.07
 -0.07
 -0.07
 -0.07
 -0.07
 -0.07
 -0.08
 -0.08
 -0.08
 -0.08
 -0.08
 -0.08
 -0.09
 -0.09
 -0.09
 -0.09
 -0.09
 -0.09
 -0.09
 -0.09
 -0.09
 -0.09
 -0.09
 -0.09
 -0.09
 -0.10
 -0.10
 -0.10
 -0.10
 -0.10
 -0.10
 -0.10
 -0.10
 -0.10
 -0.10
 -0.10
  101  Proteins eluted with mild detergent and salt (weak parkin interactors – CCCP conditions) LOC389901 hypothetical LOC389901
 SCYE1 Multisynthetase complex auxiliary component p43
 GLUD1 GLUD1 protein
 SEC62 Translocation protein SEC62
 CDC5L Cell division cycle 5-like protein
 MRPL12 39S ribosomal protein L12, mitochondrial
 NCL Isoform 2 of Nucleolin
 NCL Isoform 2 of Nucleolin
 DLAT Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial
 NDUFA8 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 8
 VARS Valyl-tRNA synthetase
 HIST1H3F;HIST1H3B;HIST1H3D;HIST1H3A;HIST1H3H;HIST1H3I;HIST1H3E;HIST1H3C;HIST1 H3J;HIST1H3G;HIST1H2BN Histone H3.1
 ALCAM 62 kDa protein
 AKAP12 Isoform 1 of A-kinase anchor protein 12
 KCTD12 BTB/POZ domain-containing protein KCTD12
 SPAG9 Isoform 4 of C-jun-amino-terminal kinase-interacting protein 4
 RIMS1 Isoform 1 of Regulating synaptic membrane exocytosis protein 1
 PSMB5 29 kDa protein
 TPM3 tropomyosin 3 isoform 4
 EIF4H Isoform Long of Eukaryotic translation initiation factor 4H
 TPM4 Isoform 1 of Tropomyosin alpha-4 chain
 ATP5C1 Isoform Heart of ATP synthase subunit gamma, mitochondrial
 IMPDH1 inosine monophosphate dehydrogenase 1 isoform a
 RPS25 40S ribosomal protein S25
 EEF1G Elongation factor 1-gamma
 SLC25A5 ADP/ATP translocase 2
 DPYSL2 Dihydropyrimidinase-related protein 2
 PCMT1 Isoform 1 of Protein-L-isoaspartate(D-aspartate) O-methyltransferase
 GANAB Isoform 1 of Neutral alpha-glucosidase AB
 TUBB Tubulin beta chain
 TUBB Tubulin beta chain
 TUBB Tubulin beta chain
 PUS7 Pseudouridylate synthase 7 homolog
 SEC63 Translocation protein SEC63 homolog
 DDOST dolichyl-diphosphooligosaccharide-protein glycosyltransferase precursor
 NT5DC1 5'-nucleotidase domain-containing protein 1
 RPSA 33 kDa protein
 EIF3I Eukaryotic translation initiation factor 3 subunit I
 CNTN1 Isoform 1 of Contactin-1
 OGT Isoform 3 of UDP-N-acetylglucosamine--peptide N-acetylglucosaminyltransferase 110 kDa subunit
 HNRNPU Isoform Short of Heterogeneous nuclear ribonucleoprotein U
 GPHN Isoform 1 of Gephyrin
 SMCHD1 Isoform 1 of Structural maintenance of chromosomes flexible hinge domain-containing protein 1
 GOT1 Aspartate aminotransferase, cytoplasmic
 LRPPRC Leucine-rich PPR motif-containing protein, mitochondrial
 CCT2 T-complex protein 1 subunit beta
 C1QBP Complement component 1 Q subcomponent-binding protein, mitochondrial
 ATP1A1 Isoform Long of Sodium/potassium-transporting ATPase subunit alpha-1
 HSPA4 Heat shock 70 kDa protein 4
 BSG Isoform 2 of Basigin
 IMPDH2 Inosine-5'-monophosphate dehydrogenase 2
 C1orf115 Uncharacterized protein C1orf115
 SEPT7 Isoform 1 of Septin-7
 RPS7 40S ribosomal protein S7
 PSMA6 Similar to Prosomal P27K protein
 DIAPH1 diaphanous 1 isoform 2
 PSMD14 26S proteasome non-ATPase regulatory subunit 14
 GTF2A1 Isoform 42 kDa of Transcription initiation factor IIA subunit 1
 ZNF207 Isoform 1 of Zinc finger protein 207
 ACTA2 Actin, aortic smooth muscle
 SEPT9 Isoform 1 of Septin-9
 PHB Prohibitin
 PSMB6 Proteasome subunit beta type-6
 CCT7 T-complex protein 1 subunit eta
 SCRN1 Secernin-1
 ENAH Isoform 2 of Protein enabled homolog
 EIF2A Eukaryotic translation initiation factor 2A
 ARPC3 Actin-related protein 2/3 complex subunit 3
 PPP2R2C Serine/threonine-protein phosphatase 2A 55 kDa regulatory subunit B gamma isoform
 HNRNPK Isoform 1 of Heterogeneous nuclear ribonucleoprotein K
 RPS5 40S ribosomal protein S5
 RARS Isoform Complexed of Arginyl-tRNA synthetase, cytoplasmic
 MAPRE1 Microtubule-associated protein RP/EB family member 1
 VPS13D Isoform 2 of Vacuolar protein sorting-associated protein 13D
 HSPA9 Stress-70 protein, mitochondrial
 HSPA9 Stress-70 protein, mitochondrial
 HSPA1L Heat shock 70kDa protein 1-like variant
  Protein Mascot Score  # of peptides  log2(heavy /light)  510
 186
 537
 70
 33
 33
 3694
 3694
  10
 5
 11
 1
 1
 1
 70
 70
  -0.10
 -0.11
 -0.11
 -0.11
 -0.11
 -0.11
 -0.11
 -0.11
  283
 104
 1662
  7
 3
 26
  -0.11
 -0.11
 -0.11
  311
 679
 338
 250
 152
 27
 245
 873
 262
 1341
 205
 91
 254
 756
 184
 3108
 583
 704
 13048
 13048
 13048
 293
 36
 255
 40
 1836
 835
 87
  8
 11
 6
 4
 4
 1
 5
 13
 5
 24
 4
 2
 5
 14
 3
 48
 9
 15
 217
 217
 217
 6
 1
 6
 1
 30
 15
 2
  -0.11
 -0.12
 -0.12
 -0.12
 -0.12
 -0.12
 -0.12
 -0.12
 -0.12
 -0.13
 -0.13
 -0.13
 -0.13
 -0.13
 -0.13
 -0.13
 -0.13
 -0.13
 -0.13
 -0.13
 -0.13
 -0.13
 -0.13
 -0.14
 -0.14
 -0.14
 -0.14
 -0.14
  68
 2307
 40
  2
 44
 1
  -0.14
 -0.14
 -0.14
  29
 26
 1269
 848
 902
 642
 624
 279
 683
 25
 882
 530
 408
 47
 160
 61
 29
 231
 626
 166
 31
 1145
 210
 123
 59
 43
 25
 2880
 214
 148
 161
 26
 3083
 3083
 265
  1
 1
 23
 13
 14
 8
 11
 6
 12
 1
 13
 8
 5
 1
 3
 1
 1
 6
 15
 2
 1
 21
 4
 3
 1
 1
 1
 50
 3
 4
 4
 1
 53
 53
 4
  -0.14
 -0.15
 -0.15
 -0.15
 -0.15
 -0.15
 -0.15
 -0.15
 -0.15
 -0.15
 -0.15
 -0.15
 -0.15
 -0.15
 -0.15
 -0.15
 -0.15
 -0.16
 -0.16
 -0.16
 -0.16
 -0.16
 -0.16
 -0.16
 -0.16
 -0.16
 -0.16
 -0.16
 -0.16
 -0.16
 -0.16
 -0.16
 -0.16
 -0.16
 -0.17
  102  Proteins eluted with mild detergent and salt (weak parkin interactors – CCCP conditions) PRKDC Isoform 1 of DNA-dependent protein kinase catalytic subunit
 DNM2 Dynamin 2 isoform 4 variant (Fragment)
 CCT4 T-complex protein 1 subunit delta
 DUT 24 kDa protein
 VPS35 Vacuolar protein sorting-associated protein 35
 PDIA6 Isoform 2 of Protein disulfide-isomerase A6
 HIST2H2AA4;HIST2H2AA3 Histone H2A type 2-A
 ERP29 Endoplasmic reticulum protein ERp29
 SET Isoform 1 of Protein SET
 TROVE2 Isoform Long of 60 kDa SS-A/Ro ribonucleoprotein
 GSR Isoform Mitochondrial of Glutathione reductase, mitochondrial
 JTV1 Multisynthetase complex auxiliary component p38
 NPM1 Isoform 1 of Nucleophosmin
 NPM1 Isoform 1 of Nucleophosmin
 PRKCSH Glucosidase 2 subunit beta
 UBE2I SUMO-conjugating enzyme UBC9
 GLO1 Lactoylglutathione lyase
 SNRPD3 Small nuclear ribonucleoprotein Sm D3
 SRPK1 Isoform 1 of Serine/threonine-protein kinase SRPK1
 SRM Spermidine synthase
 SFRS3 Splicing factor, arginine/serine-rich 3
 MARCKS Myristoylated alanine-rich C-kinase substrate
 GCN1L1 Translational activator GCN1
 PODXL2 Isoform 2 of Podocalyxin-like protein 2
 RPL23 Similar to ribosomal protein L23
 EEF1D eukaryotic translation elongation factor 1 delta isoform 1
 - Farnesyl pyrophosphate synthetase like-4 protein (Fragment)
 DCTN2 dynactin 2
 RPL18 60S ribosomal protein L18
 CCT8 59 kDa protein
 AP2M1 Isoform 1 of AP-2 complex subunit mu-1
 HSPA8 Isoform 1 of Heat shock cognate 71 kDa protein
 HSPA8 Isoform 1 of Heat shock cognate 71 kDa protein
 SPTAN1 Isoform 2 of Spectrin alpha chain, brain
 WBP2 WW domain-binding protein 2
 U2AF2 Splicing factor U2AF 65 kDa subunit
 OCIAD1 Isoform 2 of OCIA domain-containing protein 1
 PGM1 Isoform 1 of Phosphoglucomutase-1
 RPL27A 60S ribosomal protein L27a
 ACTR2 Actin-related protein 2
 CALR Calreticulin
 UQCRC2 Cytochrome b-c1 complex subunit 2, mitochondrial
 SND1 Staphylococcal nuclease domain-containing protein 1
 C14orf156 SRA stem-loop-interacting RNA-binding protein, mitochondrial
 PDIA3 14 kDa protein
 CSE1L Isoform 1 of Exportin-2
 SF3A3 Splicing factor 3A subunit 3
 - Similar to Mitochondrial ribosomal protein L38
 COPS4 COP9 signalosome complex subunit 4
 RPLP2 60S acidic ribosomal protein P2
 PSMD13 proteasome 26S non-ATPase subunit 13 isoform 2
 NCBP1 Nuclear cap-binding protein subunit 1
 CIP29 18 kDa protein
 YBX1 Nuclease-sensitive element-binding protein 1
 FKBP4 FK506-binding protein 4
 PFAS Phosphoribosylformylglycinamidine synthase
 CCDC25 5 kDa protein
 ARID4A Isoform III of AT-rich interactive domain-containing protein 4A
 - cDNA FLJ46890 fis, clone UTERU3018172
 TUBA1B Tubulin alpha-1B chain
 TUBA1B Tubulin alpha-1B chain
 PAFAH1B1 Uncharacterized protein PAFAH1B1 (Fragment)
 GNB1 Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
 CNN3 Calponin-3
 LRRC59 Leucine-rich repeat-containing protein 59
 TLE1 Transducin-like enhancer protein 1
 VDAC1 Voltage-dependent anion-selective channel protein 1
 SORD Sorbitol dehydrogenase
 HN1 Isoform 1 of Hematological and neurological expressed 1 protein
 ECH1 Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase, mitochondrial
 RPS2 40S ribosomal protein S2
 SF3B5 Splicing factor 3B subunit 5
 VBP1 Von Hippel-Lindau binding protein 1
 RPS21 40S ribosomal protein S21
 PPIB peptidylprolyl isomerase B precursor
 RPS6 40S ribosomal protein S6
 NDUFS4 NADH dehydrogenase [ubiquinone] iron-sulfur protein 4, mitochondrial
 SMARCC1 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily c, member 1
 RNF130 Goliath homolog
 HK1 Isoform 1 of Hexokinase-1
  Protein Mascot Score  # of peptides  log2(heavy /light)  802
 83
 1114
 574
 102
 1065
 661
 116
 863
 308
 88
 54
 3159
 3159
 608
 57
 294
 25
 27
 37
 526
 342
 62
 27
 944
 877
 66
 345
 326
 837
 29
 6557
 6557
 821
 29
 511
 27
 26
 276
 68
 1113
 248
 701
 166
 79
 352
 84
 38
 30
 527
 247
 150
 35
 1221
 611
 211
 57
 27
 26
 7000
 7000
 103
 411
 252
 131
 32
 1490
 1004
 265
 109
 478
 173
 98
 482
 312
 290
 44
  19
 3
 21
 7
 2
 18
 14
 1
 16
 5
 2
 1
 51
 51
 11
 1
 6
 1
 1
 1
 11
 5
 2
 1
 13
 17
 2
 6
 6
 16
 1
 111
 111
 14
 1
 8
 1
 1
 8
 2
 23
 6
 12
 3
 1
 7
 2
 1
 1
 9
 4
 4
 1
 18
 12
 5
 2
 1
 1
 126
 126
 2
 9
 5
 3
 1
 28
 20
 5
 2
 10
 3
 3
 8
 6
 7
 1
  -0.17
 -0.17
 -0.17
 -0.17
 -0.17
 -0.17
 -0.17
 -0.17
 -0.17
 -0.17
 -0.17
 -0.17
 -0.17
 -0.17
 -0.18
 -0.18
 -0.18
 -0.18
 -0.18
 -0.18
 -0.18
 -0.18
 -0.18
 -0.18
 -0.18
 -0.18
 -0.18
 -0.19
 -0.19
 -0.19
 -0.19
 -0.19
 -0.19
 -0.19
 -0.19
 -0.19
 -0.19
 -0.19
 -0.20
 -0.20
 -0.20
 -0.20
 -0.20
 -0.20
 -0.20
 -0.20
 -0.20
 -0.20
 -0.20
 -0.20
 -0.20
 -0.20
 -0.20
 -0.21
 -0.21
 -0.21
 -0.21
 -0.21
 -0.21
 -0.21
 -0.21
 -0.21
 -0.21
 -0.21
 -0.21
 -0.21
 -0.21
 -0.21
 -0.21
 -0.21
 -0.22
 -0.22
 -0.22
 -0.22
 -0.22
 -0.22
 -0.22
  130
 25
 439
  4
 1
 8
  -0.22
 -0.22
 -0.22
  103  Proteins eluted with mild detergent and salt (weak parkin interactors – CCCP conditions) - Uncharacterized protein ENSP00000348237
 DHX15 Putative pre-mRNA-splicing factor ATP-dependent RNA helicase DHX15
 HADH Isoform 1 of Hydroxyacyl-coenzyme A dehydrogenase, mitochondrial
 RPL3 60S ribosomal protein L3
 RPL27 60S ribosomal protein L27
 CCT3 chaperonin containing TCP1, subunit 3 isoform b
 MDH2 Malate dehydrogenase, mitochondrial
 GBAS Protein NipSnap homolog 2
 UBA1 Ubiquitin-like modifier-activating enzyme 1
 SNRPB Isoform SM-B' of Small nuclear ribonucleoprotein-associated proteins B and B'
 GOT2 Aspartate aminotransferase, mitochondrial
 GNG4 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-4
 RPL8 60S ribosomal protein L8
 RPL30 60S ribosomal protein L30
 NUDC Nuclear migration protein nudC
 LARP1 Isoform 3 of La-related protein 1
 HNRNPAB Isoform 2 of Heterogeneous nuclear ribonucleoprotein A/B
 CAP1 Adenylyl cyclase-associated protein 1
 ALDH18A1 Isoform Long of Delta-1-pyrroline-5-carboxylate synthetase
 ATP6V1B2 Vacuolar ATP synthase subunit B, brain isoform
 NLN cDNA FLJ14696 fis, clone NT2RP2005775, highly similar to NEUROLYSIN
 TAGLN2 24 kDa protein
 SGTA Small glutamine-rich tetratricopeptide repeat-containing protein alpha
 ARD1A N-terminal acetyltransferase complex ARD1 subunit homolog A
 DLD Dihydrolipoyl dehydrogenase, mitochondrial
 SNRPD1 Small nuclear ribonucleoprotein Sm D1
 CS citrate synthase precursor, isoform b
 GLT25D1 Glycosyltransferase 25 family member 1
 MAPK1IP1L MAPK-interacting and spindle-stabilizing protein-like
 GPSN2 Isoform 1 of Synaptic glycoprotein SC2
 CDC42 Isoform 2 of Cell division control protein 42 homolog
 BPNT1 Isoform 1 of 3'(2'),5'-bisphosphate nucleotidase 1
 FARSB Phenylalanyl-tRNA synthetase beta chain
 PSMC5 26S protease regulatory subunit 8
 PLD3 Phospholipase D3
 PSMC4 Isoform 1 of 26S protease regulatory subunit 6B
 GAP43 Neuromodulin
 ETFB Isoform 1 of Electron transfer flavoprotein subunit beta
 FH Isoform Mitochondrial of Fumarate hydratase, mitochondrial
 MAT2A S-adenosylmethionine synthetase isoform type-2
 FUS Isoform Short of RNA-binding protein FUS
 DCX Doublecortex
 MT-CO2 Cytochrome c oxidase subunit 2
 KHSRP Isoform 1 of Far upstream element-binding protein 2
 HINT1 Histidine triad nucleotide-binding protein 1
 CHGA chromogranin A precursor
 CALM3;CALM2;CALM1 Calmodulin
 RIOK1 Serine/threonine-protein kinase RIO1
 PSMD12 26S proteasome non-ATPase regulatory subunit 12
 BCKDHA 2-oxoisovalerate dehydrogenase subunit alpha, mitochondrial
 S100A6 Protein S100-A6
 HSP90AA1 Isoform 1 of Heat shock protein HSP 90-alpha
 OLA1 Isoform 2 of Obg-like ATPase 1
 ENO1 Isoform alpha-enolase of Alpha-enolase
 ENO1 Isoform alpha-enolase of Alpha-enolase
 - Dihydropyrimidinase-like 2 long form (Fragment)
 ILF2 Interleukin enhancer-binding factor 2
 RBM12 RNA-binding protein 12
 PDIA3 Protein disulfide-isomerase A3
 PDIA3 Protein disulfide-isomerase A3
 NONO Non-POU domain-containing octamer-binding protein
 G3BP1 Ras GTPase-activating protein-binding protein 1
 RPL7 60S ribosomal protein L7
 PRDX1 Peroxiredoxin-1
 NANS Sialic acid synthase
 SF3B3 Isoform 1 of Splicing factor 3B subunit 3
 MTDH Protein LYRIC
  1SNRPD2 Small nuclear ribonucleoprotein Sm D2
 MRPS22 28S ribosomal protein S22, mitochondrial
 KIF5B Kinesin-1 heavy chain
 AHCY Adenosylhomocysteinase
 SFRS2 Splicing factor, arginine/serine-rich 2
 PSMC6 26S protease regulatory subunit S10B
 TIMM23 Mitochondrial import inner membrane translocase subunit Tim23
 PDCD5 Programmed cell death protein 5
 NAP1L4 Nucleosome assembly protein 1-like 4
 PFN2 Isoform IIb of Profilin-2
 TBCA Tubulin-specific chaperone A
 BIRC6 baculoviral IAP repeat-containing 6
 SAE1 SUMO-activating enzyme subunit 1
  Protein Mascot Score  # of peptides  log2(heavy /light)  847
 530
 331
 321
 153
 2519
 1892
 36
 2935
 131
 1097
 29
 795
 268
 205
 105
 667
 545
 329
 237
 58
 893
 192
 31
 514
 399
 215
 188
 67
 60
 35
 31
 184
 384
 57
 115
 128
 32
 1283
 190
 272
 177
 27
 591
 168
 247
 207
 58
 80
 66
 237
 2330
 37
 6222
 6222
 185
 121
 47
 2852
 2852
 2632
 315
 255
 1084
 439
 108
 85
  14
 9
 6
 6
 4
 44
 32
 1
 44
 3
 21
 1
 16
 5
 3
 3
 11
 10
 7
 5
 1
 13
 4
 1
 9
 7
 6
 4
 1
 1
 1
 1
 5
 7
 2
 3
 4
 1
 21
 3
 6
 4
 1
 14
 4
 6
 5
 2
 2
 2
 3
 46
 1
 97
 97
 3
 3
 1
 52
 52
 46
 5
 7
 27
 9
 3
 2
  -0.22
 -0.22
 -0.22
 -0.22
 -0.22
 -0.22
 -0.22
 -0.22
 -0.23
 -0.23
 -0.23
 -0.23
 -0.23
 -0.23
 -0.23
 -0.23
 -0.24
 -0.24
 -0.24
 -0.24
 -0.24
 -0.24
 -0.24
 -0.24
 -0.24
 -0.24
 -0.24
 -0.24
 -0.24
 -0.24
 -0.24
 -0.24
 -0.24
 -0.24
 -0.24
 -0.25
 -0.25
 -0.25
 -0.25
 -0.25
 -0.25
 -0.25
 -0.25
 -0.26
 -0.26
 -0.26
 -0.26
 -0.26
 -0.26
 -0.26
 -0.26
 -0.26
 -0.26
 -0.26
 -0.26
 -0.27
 -0.27
 -0.27
 -0.27
 -0.27
 -0.27
 -0.27
 -0.27
 -0.27
 -0.27
 -0.27
 -0.27
  221
 130
 550
 535
 288
 461
 32
 91
 271
 59
 36
 26
 25
  6
 2
 10
 11
 5
 11
 1
 2
 4
 1
 1
 1
 1
  -0.27
 -0.27
 -0.28
 -0.28
 -0.28
 -0.28
 -0.28
 -0.28
 -0.28
 -0.28
 -0.28
 -0.28
 -0.28
  104  Proteins eluted with mild detergent and salt (weak parkin interactors – CCCP conditions) IDH2 Isocitrate dehydrogenase [NADP], mitochondrial
 PPA1 Inorganic pyrophosphatase
 EIF2S3 Eukaryotic translation initiation factor 2 subunit 3
 PTPLAD1 Protein tyrosine phosphatase-like protein PTPLAD1
 KPNB1 Importin subunit beta-1
 IDH1 Isocitrate dehydrogenase [NADP] cytoplasmic
 PSMD1 Isoform 1 of 26S proteasome non-ATPase regulatory subunit 1
 SRP9 SRP9 protein
 CRMP1 Dihydropyrimidinase-related protein 1
 RPL18A 60S ribosomal protein L18a
 BCLAF1 Isoform 1 of Bcl-2-associated transcription factor 1
 EZR Ezrin
 SNRPA U1 small nuclear ribonucleoprotein A
 IARS2 Isoleucyl-tRNA synthetase, mitochondrial
 ANP32B Isoform 1 of Acidic leucine-rich nuclear phosphoprotein 32 family member B
 PAICS Multifunctional protein ADE2
 FAM50A Protein FAM50A
 HSP90AB1 Heat shock protein HSP 90-beta
 HSP90AB1 Heat shock protein HSP 90-beta
 TKT Transketolase
 ATP6V1E1 Vacuolar proton pump subunit E 1
 CTNNA1 Isoform 1 of Catenin alpha-1
 LOC729611 similar to hCG1641491 isoform 2
 RPA2 Isoform 1 of Replication protein A 32 kDa subunit
 SHMT2 Serine hydroxymethyltransferase, mitochondrial
 NACA Nascent polypeptide-associated complex subunit alpha
 FKBP10 CDNA: FLJ22221 fis, clone HRC01651
 VAPA Vesicle-associated membrane protein-associated protein A
 FRAS1 Isoform 2 of Extracellular matrix protein FRAS1
 PRDX5 Isoform Mitochondrial of Peroxiredoxin-5, mitochondrial
 ATP6V1A Vacuolar ATP synthase catalytic subunit A
 GAPDH Glyceraldehyde-3-phosphate dehydrogenase
 GAPDH Glyceraldehyde-3-phosphate dehydrogenase
 HSPE1 10 kDa heat shock protein, mitochondrial
 PDIA4 Protein disulfide-isomerase A4
 DCLK1 Isoform 2 of Serine/threonine-protein kinase DCLK1
 ACAA2 3-ketoacyl-CoA thiolase, mitochondrial
 TCEB1 Transcription elongation factor B polypeptide 1
 TUFM Tu translation elongation factor, mitochondrial precursor
 ANXA2 Annexin A2
 RBBP4 Histone-binding protein RBBP4
 C20orf3 Adipocyte plasma membrane-associated protein
 EIF4EBP1 Eukaryotic translation initiation factor 4E-binding protein 1
 METAP2 Methionine aminopeptidase 2
 AHCYL2 Putative adenosylhomocysteinase 3
 PRMT1 HMT1 hnRNP methyltransferase-like 2 isoform 1
 TPP2 Tripeptidyl-peptidase 2
 THY1 Thy-1 membrane glycoprotein
 MCM3 DNA replication licensing factor MCM3
 PSMC1 26S protease regulatory subunit 4
 SUGT1 Isoform 2 of Suppressor of G2 allele of SKP1 homolog
 SLC25A1 Tricarboxylate transport protein, mitochondrial
 TUBB2C Tubulin beta-2C chain
 TMPO Isoform Gamma of Lamina-associated polypeptide 2, isoforms beta/gamma
 USP14 Ubiquitin carboxyl-terminal hydrolase 14
 LOC651249 similar to Ribosomal protein L34
 RPL11 Isoform 1 of 60S ribosomal protein L11
 COX4I1 Cytochrome c oxidase subunit 4 isoform 1, mitochondrial
 PSMB3 Proteasome subunit beta type-3
 - PRO1446
 SCAPER Isoform 1 of S phase cyclin A-associated protein in the endoplasmic reticulum
 NNT NAD(P) transhydrogenase, mitochondrial
 HYOU1 Hypoxia up-regulated protein 1
 CPNE7 Isoform 1 of Copine-7
 YWHAE 14-3-3 protein epsilon
 - 12 kDa protein
 PSMD5 26S proteasome non-ATPase regulatory subunit 5
 CTTN cortactin isoform b
 RPL9 60S ribosomal protein L9
 CDC45L CDC45-related protein
 PHGDH D-3-phosphoglycerate dehydrogenase
 HYDIN2;HYDIN Uncharacterized protein HYDIN2
 CNBP Isoform 1 of Cellular nucleic acid-binding protein
 RPL14 60S ribosomal protein L14
 RPL7A 60S ribosomal protein L7a
 NP CDNA FLJ25678 fis, clone TST04067, highly similar to PURINE NUCLEOSIDE PHOSPHORYLASE
 SDHA SDHA protein
 LAP3 Isoform 1 of Cytosol aminopeptidase
 YWHAB Isoform Long of 14-3-3 protein beta/alpha
 LDHB L-lactate dehydrogenase B chain
  Protein Mascot Score  # of peptides  log2(heavy /light)  714
 361
 181
 166
 877
 385
 158
 106
 875
 220
 31
 930
 208
 30
 198
 314
 66
 6777
 6777
 1845
 200
 120
 90
 48
 628
 238
 184
 27
 52
 37
 380
 7488
 7488
 990
 358
 29
 443
 61
 1037
 467
 344
 315
 119
 83
 39
 636
 137
 39
 272
 345
 197
 27
 878
 568
 152
 219
 323
 58
 88
 28
 27
 538
 586
 28
 2113
 132
 92
 107
 189
 34
 1351
 30
 255
 220
 580
  13
 6
 3
 4
 19
 7
 3
 3
 16
 5
 1
 15
 6
 1
 4
 6
 1
 132
 132
 30
 3
 2
 2
 1
 11
 4
 3
 1
 2
 1
 9
 132
 132
 16
 9
 1
 6
 1
 17
 11
 7
 9
 3
 2
 1
 12
 4
 1
 7
 8
 4
 1
 12
 11
 3
 6
 7
 2
 3
 1
 1
 10
 11
 1
 33
 2
 1
 2
 5
 1
 25
 1
 5
 5
 10
  -0.29
 -0.29
 -0.29
 -0.29
 -0.29
 -0.29
 -0.29
 -0.29
 -0.29
 -0.29
 -0.29
 -0.29
 -0.29
 -0.29
 -0.30
 -0.30
 -0.30
 -0.30
 -0.30
 -0.30
 -0.30
 -0.30
 -0.30
 -0.30
 -0.30
 -0.30
 -0.30
 -0.30
 -0.30
 -0.30
 -0.30
 -0.30
 -0.30
 -0.31
 -0.31
 -0.31
 -0.31
 -0.31
 -0.31
 -0.31
 -0.31
 -0.31
 -0.31
 -0.31
 -0.31
 -0.31
 -0.31
 -0.31
 -0.32
 -0.32
 -0.32
 -0.32
 -0.32
 -0.32
 -0.32
 -0.32
 -0.32
 -0.32
 -0.32
 -0.32
 -0.32
 -0.32
 -0.32
 -0.32
 -0.32
 -0.32
 -0.32
 -0.32
 -0.32
 -0.32
 -0.33
 -0.33
 -0.33
 -0.33
 -0.33
  533
 27
 110
 743
 682
  12
 1
 2
 10
 17
  -0.33
 -0.33
 -0.33
 -0.33
 -0.33
  105  Proteins eluted with mild detergent and salt (weak parkin interactors – CCCP conditions)  Protein Mascot Score  # of peptides  log2(heavy /light)  RPS27 40S ribosomal protein S27
 EEF1A1 Elongation factor 1-alpha 1
 EEF1A1 Elongation factor 1-alpha 1
 GPI Glucose-6-phosphate isomerase
 SERBP1 Isoform 1 of Plasminogen activator inhibitor 1 RNA-binding protein
 ISYNA1 58 kDa protein
 YWHAG 14-3-3 protein gamma
 VAT1 Synaptic vesicle membrane protein VAT-1 homolog
 RPL12 Isoform 1 of 60S ribosomal protein L12
 RPLP1 60S acidic ribosomal protein P1
 YWHAZ 14-3-3 protein zeta/delta
 RPL13 60S ribosomal protein L13
 TOMM70A Mitochondrial import receptor subunit TOM70
 PKM2 Isoform M2 of Pyruvate kinase isozymes M1/M2
 PKM2 Isoform M2 of Pyruvate kinase isozymes M1/M2
 EIF4A1 Eukaryotic initiation factor 4A-I
 DHX9 ATP-dependent RNA helicase A
 RPLP0 60S acidic ribosomal protein P0
 TUBB2B Tubulin beta-2B chain
 HADHB Trifunctional enzyme subunit beta, mitochondrial
 RNPEP Uncharacterized protein RNPEP
 C14orf166 UPF0568 protein C14orf166
 DNM3 Isoform 1 of Dynamin-3
 PA2G4 Proliferation-associated protein 2G4
 PFN1 Profilin-1
 SYNCRIP Isoform 2 of Heterogeneous nuclear ribonucleoprotein Q
 PROSC Proline synthetase co-transcribed bacterial homolog protein
 UBE2M NEDD8-conjugating enzyme Ubc12
 - 8 kDa protein
 VGF VGF nerve growth factor inducible precursor
 PSMD3 26S proteasome non-ATPase regulatory subunit 3
 COPA Coatomer subunit alpha
 SF3B2 splicing factor 3B subunit 2
 MACROD1 MACRO domain-containing protein 1
 STIP1 STIP1 protein
 PTBP1 Isoform 1 of Polypyrimidine tract-binding protein 1
 RPL6 60S ribosomal protein L6
 SLC25A11 Mitochondrial 2-oxoglutarate/malate carrier protein
 TIPRL Isoform 2 of TIP41-like protein
 VDAC2 Isoform 3 of Voltage-dependent anion-selective channel protein 2
 ACTBL2 Beta-actin-like protein 2
 SNRP70 Isoform 2 of U1 small nuclear ribonucleoprotein 70 kDa
 IPO5 importin 5
 MCTS1 Isoform 1 of Malignant T cell amplified sequence 1
 HS6ST2 Isoform 2 of Heparan-sulfate 6-O-sulfotransferase 2
 UBA2 SUMO-activating enzyme subunit 2
 PTK7 PTK7 protein tyrosine kinase 7 isoform a variant (Fragment)
 ASAH1 Acid ceramidase
 ANKRD20A1 Ankyrin repeat domain-containing protein 20A1
 ANP32A Acidic leucine-rich nuclear phosphoprotein 32 family member A
 HSPH1 Isoform Beta of Heat shock protein 105 kDa
 DDX1 ATP-dependent RNA helicase DDX1
 CHMP4B Charged multivesicular body protein 4b
 SV2A Isoform 1 of Synaptic vesicle glycoprotein 2A
 RCC1 regulator of chromosome condensation 1 isoform a
 MTA2 Metastasis-associated protein MTA2
 NDUFS2 NADH dehydrogenase [ubiquinone] iron-sulfur protein 2, mitochondrial
 TDRKH Isoform 1 of Tudor and KH domain-containing protein
 HADHA Trifunctional enzyme subunit alpha, mitochondrial
 MTHFD1 C-1-tetrahydrofolate synthase, cytoplasmic
 RPL37A 60S ribosomal protein L37a
 ATAD3B Isoform 2 of ATPase family AAA domain-containing protein 3B
 ATP5L ATP synthase subunit g, mitochondrial
 TUBB2A Tubulin beta-2A chain
 PSMA3 Isoform 2 of Proteasome subunit alpha type-3
 EMD Emerin
 FNIP2 Isoform 1 of Folliculin-interacting protein 2
 CLTC Isoform 1 of Clathrin heavy chain 1
 GNB2 Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2
 EEF1A2 Elongation factor 1-alpha 2
 PRDX6 Peroxiredoxin-6
 RLTPR Leucine-rich repeat-containing protein 16C
 UQCRH Cytochrome b-c1 complex subunit 6, mitochondrial
 TMED10 Transmembrane emp24 domain-containing protein 10
 TUBA1A Tubulin alpha-1A chain
 PPT1 Palmitoyl-protein thioesterase 1
 L1CAM Isoform 1 of Neural cell adhesion molecule L1
 AARSD1 Alanyl-tRNA synthetase, class IIc family protein
 ARHGDIA Rho GDP-dissociation inhibitor 1
 RPL35 60S ribosomal protein L35
 MRE11A Isoform 1 of Double-strand break repair protein MRE11A
  232
 6058
 6058
 1964
 120
 36
 1277
 465
 445
 47
 826
 322
 209
 6290
 6290
 1003
 672
 467
 518
 270
 103
 79
 115
 309
 1218
 725
 32
 143
 27
 883
 314
 94
 418
 112
 573
 1571
 345
 154
 50
 742
 224
 35
 266
 58
 29
 72
 28
 30
 26
 1319
 326
 345
 34
 96
 255
 163
 110
 28
 1491
 985
 229
 82
 27
 56
 68
 36
 37
 1773
 140
 1121
 1157
 34
 130
 172
 236
 321
 120
 29
 572
 233
 80
  5
 121
 121
 35
 4
 1
 15
 8
 8
 1
 11
 7
 6
 110
 110
 19
 15
 9
 9
 7
 3
 2
 4
 8
 23
 14
 1
 3
 1
 14
 7
 3
 8
 3
 12
 27
 9
 2
 2
 15
 6
 1
 7
 2
 1
 2
 1
 1
 1
 22
 7
 7
 1
 3
 4
 4
 2
 1
 27
 18
 6
 2
 1
 2
 1
 1
 1
 31
 3
 23
 19
 1
 2
 3
 5
 3
 3
 1
 8
 6
 2
  -0.33
 -0.33
 -0.33
 -0.33
 -0.33
 -0.33
 -0.34
 -0.34
 -0.34
 -0.34
 -0.34
 -0.34
 -0.34
 -0.34
 -0.34
 -0.34
 -0.34
 -0.35
 -0.35
 -0.35
 -0.35
 -0.35
 -0.35
 -0.35
 -0.35
 -0.35
 -0.35
 -0.36
 -0.36
 -0.36
 -0.36
 -0.36
 -0.36
 -0.36
 -0.36
 -0.37
 -0.37
 -0.37
 -0.37
 -0.37
 -0.37
 -0.38
 -0.38
 -0.38
 -0.38
 -0.38
 -0.38
 -0.38
 -0.38
 -0.38
 -0.38
 -0.38
 -0.38
 -0.38
 -0.39
 -0.39
 -0.39
 -0.39
 -0.39
 -0.39
 -0.39
 -0.39
 -0.39
 -0.39
 -0.39
 -0.39
 -0.39
 -0.40
 -0.40
 -0.40
 -0.40
 -0.40
 -0.40
 -0.40
 -0.40
 -0.40
 -0.40
 -0.40
 -0.41
 -0.41
 -0.41
  106  Proteins eluted with mild detergent and salt (weak parkin interactors – CCCP conditions) CSTB Cystatin-B
 TRAP1 Heat shock protein 75 kDa, mitochondrial
 ZYX Zyxin
 RPL36 60S ribosomal protein L36
 RBP1 Retinol-binding protein I, cellular
 NDUFAF2 Mimitin, mitochondrial
 MDH1 Malate dehydrogenase, cytoplasmic
 ATPIF1 Putative uncharacterized protein DKFZp564G0422
 FEN1 Flap endonuclease 1
 PYCR1 Pyrroline-5-carboxylate reductase 1, mitochondrial
 SERPINH1 Serpin H1
 SCG3 Secretogranin-3
 HNRNPF Heterogeneous nuclear ribonucleoprotein F
 AKR1C1 Aldo-keto reductase family 1 member C1
 EIF4G1 eukaryotic translation initiation factor 4 gamma, 1 isoform 2
 ALDOC Fructose-bisphosphate aldolase C
 YARS Tyrosyl-tRNA synthetase, cytoplasmic
 ATIC Bifunctional purine biosynthesis protein PURH
 ALDH7A1 aldehyde dehydrogenase 7 family, member A1
 KARS Lysyl-tRNA synthetase
 SF3A1 Splicing factor 3 subunit 1
 MBD3 Isoform 1 of Methyl-CpG-binding domain protein 3
 CACYBP Isoform 1 of Calcyclin-binding protein
 MAP4 110 kDa protein
 PSMB4 Proteasome subunit beta type-4
 PGK1 Phosphoglycerate kinase 1
 PGK1 Phosphoglycerate kinase 1
 PPP2R1A Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform
 C7orf24 Uncharacterized protein C7orf24
 CNIH4 Isoform 1 of Protein cornichon homolog 4
 UBC;UBB;RPS27A ubiquitin and ribosomal protein S27a precursor
 CEP170 Isoform 1 of Centrosomal protein of 170 kDa
 GDI1 Rab GDP dissociation inhibitor alpha
 AARS Alanyl-tRNA synthetase, cytoplasmic
 SOD1 Superoxide dismutase [Cu-Zn]
 NOMO3;NOMO1 Nodal modulator 1
 PABPC1 Isoform 1 of Polyadenylate-binding protein 1
 ZNF217 Zinc finger protein 217
 TPI1 Isoform 1 of Triosephosphate isomerase
 COPG Coatomer subunit gamma
 HSD17B10 Isoform 1 of 3-hydroxyacyl-CoA dehydrogenase type-2
 DARS Aspartyl-tRNA synthetase, cytoplasmic
 IKBKG 29 kDa protein
 NR2C2AP 19 kDa protein
 EEF2 Elongation factor 2
 EEF2 Elongation factor 2
 TUBB4 Tubulin beta-4 chain
 EWSR1 cDNA FLJ31747 fis, clone NT2RI2007377, highly similar to RNA-BINDING PROTEIN EWS
 TPT1 TPT1 protein
 DYNC1I2 Isoform 2C of Cytoplasmic dynein 1 intermediate chain 2
 AKR1B1 Aldose reductase
 SARS Seryl-tRNA synthetase, cytoplasmic
 LARS Leucyl-tRNA synthetase, cytoplasmic
 RAD23B UV excision repair protein RAD23 homolog B
 RPS12 ribosomal protein S12
 PCNP Isoform 1 of PEST proteolytic signal-containing nuclear protein
 SPTBN1 Isoform Long of Spectrin beta chain, brain 1
 NSUN2 tRNA (cytosine-5-)-methyltransferase NSUN2
 SRP14 Signal recognition particle 14 kDa protein
 CKB Creatine kinase B-type
 CKB Creatine kinase B-type
 MIF;LOC284889 Macrophage migration inhibitory factor
 ACTL6A Isoform 1 of Actin-like protein 6A
 PPIA Peptidyl-prolyl cis-trans isomerase A
 PPIA Peptidyl-prolyl cis-trans isomerase A
 LANCL1 LanC-like protein 1
 ATP5F1 ATP synthase subunit b, mitochondrial
 HMGB1 High mobility group protein B1
 RPL19 60S ribosomal protein L19
 TNPO1 transportin 1 isoform 1
 PEPD Xaa-Pro dipeptidase
 LOC641293 Similar to 60S ribosomal protein L21
 UCHL1 Ubiquitin carboxyl-terminal hydrolase isozyme L1
 MCM6 DNA replication licensing factor MCM6
 XPO1 Exportin-1
 ACTR1A Alpha-centractin
 HNRNPUL1 Isoform 1 of Heterogeneous nuclear ribonucleoprotein U-like protein 1
 ENO2 Gamma-enolase
 TM9SF3 Transmembrane 9 superfamily member 3
 PEBP1 Phosphatidylethanolamine-binding protein 1
  Protein Mascot Score  # of peptides  log2(heavy /light)  40
 1188
 81
 98
 33
 34
 250
 94
 41
 397
 129
 116
 287
 175
 753
 148
 141
 771
 35
 479
 116
 227
 468
 281
 31
 3350
 3350
 222
 40
 30
 1296
 118
 937
 709
 944
 41
 972
 30
 1339
 82
 246
 161
 29
 33
 4184
 4184
 61
  1
 20
 2
 3
 1
 1
 5
 2
 1
 5
 2
 3
 4
 3
 13
 4
 3
 12
 1
 8
 2
 4
 11
 6
 1
 53
 53
 5
 1
 1
 30
 2
 15
 13
 19
 1
 17
 1
 24
 2
 4
 4
 1
 1
 74
 74
 1
  -0.41
 -0.41
 -0.41
 -0.41
 -0.41
 -0.42
 -0.42
 -0.42
 -0.42
 -0.42
 -0.42
 -0.43
 -0.43
 -0.43
 -0.43
 -0.43
 -0.43
 -0.43
 -0.43
 -0.44
 -0.44
 -0.44
 -0.44
 -0.44
 -0.44
 -0.44
 -0.44
 -0.45
 -0.45
 -0.45
 -0.45
 -0.45
 -0.45
 -0.45
 -0.45
 -0.45
 -0.45
 -0.45
 -0.45
 -0.45
 -0.46
 -0.46
 -0.46
 -0.46
 -0.46
 -0.46
 -0.46
  242
 124
 41
 86
 494
 1222
 495
 331
 222
 154
 115
 43
 4063
 4063
 279
 27
 3804
 3804
 28
 26
 1781
 735
 292
 65
 635
 1297
 179
 48
 28
 60
 477
 32
 1582
  5
 3
 1
 3
 9
 19
 12
 7
 6
 4
 3
 1
 65
 65
 6
 1
 74
 74
 1
 1
 30
 12
 5
 2
 9
 24
 5
 1
 1
 2
 6
 1
 24
  -0.46
 -0.46
 -0.46
 -0.46
 -0.46
 -0.46
 -0.46
 -0.46
 -0.46
 -0.46
 -0.46
 -0.46
 -0.46
 -0.46
 -0.47
 -0.47
 -0.47
 -0.47
 -0.47
 -0.47
 -0.47
 -0.47
 -0.47
 -0.47
 -0.48
 -0.48
 -0.48
 -0.48
 -0.48
 -0.48
 -0.48
 -0.48
 -0.48
  107  Proteins eluted with mild detergent and salt (weak parkin interactors – CCCP conditions) FHL1 Four and a half LIM domains 1 variant
 UBQLN1 Isoform 2 of Ubiquilin-1
 USO1 Putative uncharacterized protein DKFZp451D234
 C14orf2 6.8 kDa mitochondrial proteolipid
 ABCC8 Uncharacterized protein ABCC8
 EPRS Bifunctional aminoacyl-tRNA synthetase
 RPL10A 60S ribosomal protein L10a
 S100A11 Protein S100-A11
 CAPZA2 F-actin-capping protein subunit alpha-2
 SMC4 Isoform 2 of Structural maintenance of chromosomes protein 4
 ST13 Hsc70-interacting protein
 RPL26L1 60S ribosomal protein L26-like 1
 PARK7 Protein DJ-1
 HMGB3 High mobility group protein B3
 OXCT1 Succinyl-CoA:3-ketoacid-coenzyme A transferase 1, mitochondrial
 ABCA13 Isoform 1 of ATP-binding cassette sub-family A member 13
 FSCN1 Fascin
 HMGB2 High mobility group protein B2
 RPN2 Ribophorin II
 ACSL4 Isoform Long of Long-chain-fatty-acid--CoA ligase 4
 PPM1G Protein phosphatase 1G
 ASPM Isoform 1 of Abnormal spindle-like microcephaly-associated protein
 HDGF Hepatoma-derived growth factor
 CTSB Cathepsin B
 UBE2L3 Ubiquitin-conjugating enzyme E2 L3
 C14orf102 hypothetical protein LOC55051 isoform 2
 CLIC4 Chloride intracellular channel protein 4
 ATP5O ATP synthase subunit O, mitochondrial
 DNA2 DNA replication helicase 2 homolog
 ACOT7 Isoform 1 of Cytosolic acyl coenzyme A thioester hydrolase
 DBNL Isoform 3 of Drebrin-like protein
 HNRNPD Isoform 1 of Heterogeneous nuclear ribonucleoprotein D0
 RANGAP1 Ran GTPase-activating protein 1
 EML4 EML4 protein
 ABCF1 Isoform 2 of ATP-binding cassette sub-family F member 1
 RAD23A UV excision repair protein RAD23 homolog A
 TFG Protein TFG
 LONP1 Lon protease homolog, mitochondrial
 RPL24 19 kDa protein
 CBS Isoform 1 of Cystathionine beta-synthase
 AHSA1 Activator of 90 kDa heat shock protein ATPase homolog 1
 CSNK2B;LY6G5B Casein kinase II subunit beta
 HERC2 Putative uncharacterized protein DKFZp547P028
 NXF1 Nuclear RNA export factor 1
 CD2AP CD2-associated protein
 PAFAH1B3 Platelet-activating factor acetylhydrolase IB subunit gamma
 TPD52L2 Isoform 2 of Tumor protein D54
 SCT Secretin
 EIF5A Isoform 2 of Eukaryotic translation initiation factor 5A-1
 LOC554235 Putative L-aspartate dehydrogenase
 PRPF19 Pre-mRNA-processing factor 19
 LASP1 Isoform 1 of LIM and SH3 domain protein 1
 PSAT1 Isoform 1 of Phosphoserine aminotransferase
 SMC1A Structural maintenance of chromosomes protein 1A
 SSB Lupus La protein
 GSTP1 Glutathione S-transferase P
 SF3B1 Splicing factor 3B subunit 1
 DNAJA2 DnaJ homolog subfamily A member 2
 AIFM1 Isoform 1 of Apoptosis-inducing factor 1, mitochondrial
 MAPT Isoform Tau-B of Microtubule-associated protein tau
 NOLC1 Isoform Beta of Nucleolar phosphoprotein p130
 PRDX3 Thioredoxin-dependent peroxide reductase, mitochondrial
 FABP5;FABP5L7 Fatty acid-binding protein, epidermal
 RNMTL1 RNA methyltransferase-like protein 1
 KHDRBS1 Isoform 1 of KH domain-containing, RNA-binding, signal transduction-associated protein 1
 C22orf32 Similar to Cullin-associated NEDD8-dissociated protein 1
 TIMM44 Mitochondrial import inner membrane translocase subunit TIM44
 RAB1A Isoform 1 of Ras-related protein Rab-1A
 SMS Isoform 1 of Spermine synthase
 CTPS CTP synthase 1
 PRPF31 Isoform 2 of U4/U6 small nuclear ribonucleoprotein Prp31
 DDX39 DDX39 protein
 DDX3Y;LOC100130220 ATP-dependent RNA helicase DDX3Y
 SF3B4 Splicing factor 3B subunit 4
 HSPA1B;HSPA1A Heat shock 70 kDa protein 1
 WARS Tryptophanyl-tRNA synthetase, cytoplasmic
 PHACTR2 Isoform 1 of Phosphatase and actin regulator 2
 STMN1 Stathmin
 PMPCB Mitochondrial-processing peptidase subunit beta
 TSPYL5 Testis-specific Y-encoded-like protein 5
  Protein Mascot Score  # of peptides  log2(heavy /light)  421
 138
 51
 31
 26
 791
 149
 48
 107
 66
 408
 151
 208
 76
 53
 25
 2121
 806
 411
 106
 135
 25
 502
 387
 170
 34
 140
 59
 25
 473
 57
 678
 56
 409
 265
 37
 94
 565
 246
 151
 28
 31
 29
 28
 34
 233
 95
 26
 658
 34
 130
 75
 392
 126
 183
 811
 337
 84
 394
 255
 39
 203
 160
 27
  10
 3
 1
 1
 1
 19
 3
 1
 3
 2
 9
 3
 5
 1
 1
 1
 36
 18
 6
 1
 4
 1
 11
 5
 3
 1
 3
 2
 1
 9
 2
 14
 1
 6
 4
 1
 3
 12
 5
 4
 1
 1
 1
 1
 1
 5
 2
 1
 17
 1
 2
 2
 9
 3
 5
 9
 8
 2
 8
 6
 1
 4
 5
 1
  -0.48
 -0.48
 -0.48
 -0.48
 -0.48
 -0.49
 -0.49
 -0.49
 -0.49
 -0.49
 -0.49
 -0.49
 -0.49
 -0.49
 -0.49
 -0.49
 -0.49
 -0.49
 -0.49
 -0.49
 -0.49
 -0.49
 -0.50
 -0.50
 -0.50
 -0.51
 -0.51
 -0.51
 -0.51
 -0.51
 -0.51
 -0.52
 -0.52
 -0.52
 -0.52
 -0.52
 -0.52
 -0.52
 -0.52
 -0.52
 -0.52
 -0.52
 -0.52
 -0.52
 -0.53
 -0.53
 -0.53
 -0.53
 -0.54
 -0.54
 -0.54
 -0.54
 -0.54
 -0.54
 -0.54
 -0.55
 -0.55
 -0.55
 -0.55
 -0.55
 -0.55
 -0.55
 -0.56
 -0.56
  303
 178
 33
 61
 31
 117
 44
 227
 200
 125
 402
 280
 33
 543
 36
 32
  8
 4
 1
 1
 1
 3
 1
 4
 4
 2
 8
 5
 1
 14
 1
 1
  -0.57
 -0.57
 -0.57
 -0.57
 -0.57
 -0.57
 -0.57
 -0.58
 -0.58
 -0.58
 -0.58
 -0.58
 -0.58
 -0.58
 -0.58
 -0.58
  108  Proteins eluted with mild detergent and salt (weak parkin interactors – CCCP conditions) RAN GTP-binding nuclear protein Ran
 CTSD Cathepsin D
 ASS1 Argininosuccinate synthase
 SLC25A13 Mitochondrial aspartate-glutamate carrier protein
 ASNS Asparagine synthetase [glutamine-hydrolyzing]
 CAMK4 Calcium/calmodulin-dependent protein kinase type IV
 MSH2 DNA mismatch repair protein Msh2
 NUCB1 Nucleobindin-1
 PCNA Proliferating cell nuclear antigen
 SFRS6 Isoform SRP55-1 of Splicing factor, arginine/serine-rich 6
 TBCB Tubulin folding cofactor B
 MCM4 DNA replication licensing factor MCM4
 LOC284393 similar to QM protein isoform 1
 SEC24C Protein transport protein Sec24C
 TPR nuclear pore complex-associated protein TPR
 UBE2V2 Ubiquitin-conjugating enzyme E2 variant 2
 GAB2 Isoform 1 of GRB2-associated-binding protein 2
 GSPT1 Eukaryotic peptide chain release factor GTP-binding subunit ERF3A
 PDHA1 Mitochondrial PDHA1
 ATP6V1H Isoform 1 of Vacuolar proton pump subunit H
 FRYL Isoform 2 of Protein furry homolog-like
 HNRNPA1 Isoform A1-B of Heterogeneous nuclear ribonucleoprotein A1
 SEC31A Isoform 3 of Protein transport protein Sec31A
 HNRNPL heterogeneous nuclear ribonucleoprotein L isoform b
 DNAJC7 DnaJ homolog subfamily C member 7
 ARS2 Isoform 2 of Arsenite-resistance protein 2
 MFAP5 Microfibrillar-associated protein 5
 NUP93 Nuclear pore complex protein Nup93
 RBM8A Isoform 1 of RNA-binding protein 8A
 CORO1A Coronin-1A
 LMNA Isoform A of Lamin-A/C
 PCK2 Phosphoenolpyruvate carboxykinase [GTP], mitochondrial
 TCEB2 Transcription elongation factor B polypeptide 2
 SLC1A5 Neutral amino acid transporter B(0)
 SSBP1 Single-stranded DNA-binding protein, mitochondrial
 RPL5 60S ribosomal protein L5
 ATP5J ATP synthase-coupling factor 6, mitochondrial
 ITPA Inosine triphosphate pyrophosphatase
 SFRS1 Isoform ASF-1 of Splicing factor, arginine/serine-rich 1
 TOMM34 Mitochondrial import receptor subunit TOM34
 CCAR1 Cell division cycle and apoptosis regulator protein 1
 C19orf10 UPF0556 protein C19orf10
 TMED2 Transmembrane emp24 domain-containing protein 2
 ACO2 Aconitate hydratase, mitochondrial
 CDC37 Hsp90 co-chaperone Cdc37
 PSME1 Proteasome activator complex subunit 1
 PCBP2 poly(rC) binding protein 2 isoform b
 ASNA1 Arsenical pump-driving ATPase
 NASP Isoform 1 of Nuclear autoantigenic sperm protein
 USP5 Isoform Long of Ubiquitin carboxyl-terminal hydrolase 5
 PSMD7 26S proteasome non-ATPase regulatory subunit 7
 GART Isoform Short of Trifunctional purine biosynthetic protein adenosine-3
 MCM7 Isoform 2 of DNA replication licensing factor MCM7
 ATP5I ATP synthase, H+ transporting, mitochondrial F0 complex, subunit E
 ETFA Electron transfer flavoprotein subunit alpha, mitochondrial
 HDLBP Vigilin
 CPNE1 Copine I
 HNRNPA3 Isoform 1 of Heterogeneous nuclear ribonucleoprotein A3
 KIAA1967 Isoform 1 of Protein KIAA1967
 MCM2 DNA replication licensing factor MCM2
 NMT1 Isoform Short of Glycylpeptide N-tetradecanoyltransferase 1
 U2AF1 Splicing factor U2AF 35 kDa subunit
 RPL4 60S ribosomal protein L4
 C3orf60 Uncharacterized protein C3orf60
 GAPVD1 Isoform 6 of GTPase-activating protein and VPS9 domain-containing protein 1
 PACSIN2 Isoform 1 of Protein kinase C and casein kinase substrate in neurons protein 2
 RAPGEF2 Rap guanine nucleotide exchange factor 2
 PHYHIPL Isoform 1 of Phytanoyl-CoA hydroxylase-interacting protein-like
 PTRH2 Peptidyl-tRNA hydrolase 2, mitochondrial
 FKBP3 FK506-binding protein 3
 TXN Thioredoxin
 BASP1 Brain acid soluble protein 1
 CKM Creatine kinase M-type
 HNRNPA2B1 Isoform B1 of Heterogeneous nuclear ribonucleoproteins A2/B1
 FERMT2 Isoform 1 of Fermitin family homolog 2
 TXNL1 Thioredoxin-like protein 1
 EHD1 EH domain-containing protein 1
 C1orf55 Isoform 1 of Uncharacterized protein C1orf55
 ACADVL Isoform 1 of Very long-chain specific acyl-CoA dehydrogenase, mitochondrial
 PIR Pirin
 LSM14B Isoform 1 of LSM14 protein homolog B
  Protein Mascot Score  # of peptides  log2(heavy /light)  342
 32
 770
 115
 84
 62
 36
 28
 802
 38
 184
 154
 706
 44
 27
 99
 58
 85
 62
 31
 29
 1312
 149
 907
 121
 93
 25
 141
 71
 33
 1700
 323
 177
 183
 115
 491
 85
 38
 102
 29
 40
 31
 61
 290
 300
 117
 445
 51
 161
 167
 77
 98
 30
 75
 734
 139
 65
 343
 195
 169
 26
 324
 401
 35
 56
 25
 27
 31
 53
 42
 76
 574
 149
 1803
 192
 108
 26
 26
 37
 80
 74
  7
 1
 19
 2
 2
 1
 1
 1
 11
 1
 4
 5
 15
 1
 1
 2
 2
 2
 2
 1
 1
 23
 3
 15
 2
 2
 1
 3
 2
 1
 31
 5
 3
 3
 2
 11
 2
 1
 3
 1
 1
 1
 1
 5
 3
 3
 8
 1
 3
 5
 2
 3
 1
 2
 6
 3
 2
 3
 3
 3
 1
 5
 9
 1
 2
 1
 1
 1
 1
 1
 2
 10
 3
 23
 3
 3
 1
 1
 1
 2
 2
  -0.59
 -0.59
 -0.59
 -0.59
 -0.59
 -0.60
 -0.60
 -0.60
 -0.60
 -0.60
 -0.60
 -0.60
 -0.61
 -0.61
 -0.61
 -0.62
 -0.62
 -0.62
 -0.62
 -0.62
 -0.62
 -0.62
 -0.62
 -0.63
 -0.63
 -0.63
 -0.63
 -0.64
 -0.64
 -0.64
 -0.64
 -0.64
 -0.64
 -0.64
 -0.64
 -0.65
 -0.65
 -0.65
 -0.65
 -0.65
 -0.65
 -0.66
 -0.66
 -0.66
 -0.66
 -0.67
 -0.67
 -0.67
 -0.67
 -0.68
 -0.68
 -0.68
 -0.68
 -0.68
 -0.69
 -0.69
 -0.69
 -0.69
 -0.69
 -0.69
 -0.69
 -0.70
 -0.70
 -0.70
 -0.70
 -0.70
 -0.71
 -0.71
 -0.71
 -0.72
 -0.72
 -0.73
 -0.73
 -0.75
 -0.75
 -0.76
 -0.76
 -0.76
 -0.77
 -0.77
 -0.77
  109  Proteins eluted with mild detergent and salt (weak parkin interactors – CCCP conditions) SRP72 Signal recognition particle 72 kDa protein
 PABPC4 Isoform 1 of Polyadenylate-binding protein 4
 BMP2K Isoform 3 of BMP-2-inducible protein kinase
 ATXN10 Ataxin-10
 PSMD6 17 kDa protein
 ARRB1 Isoform 1A of Beta-arrestin-1
 GDI2 Rab GDP dissociation inhibitor beta
 HNRNPH1 Heterogeneous nuclear ribonucleoprotein H
 DNAJA1 DnaJ homolog subfamily A member 1
 FKBP5 FK506-binding protein 5
 NT5E 5'-nucleotidase
 CLIC1 Chloride intracellular channel protein 1
 PRPF8 Pre-mRNA-processing-splicing factor 8
 PCBP1 Poly(rC)-binding protein 1
 MCM5 DNA replication licensing factor MCM5
 RIOK1 RIO kinase 1 isoform 2
 NLN Neurolysin, mitochondrial
 SNX6 sorting nexin 6 isoform a
 MTPN Myotrophin
 GTF2I Isoform 1 of General transcription factor II-I
 PAK3 Isoform 2 of Serine/threonine-protein kinase PAK 3
 IPO4 IPO4 protein variant (Fragment)
 VAPA 14 kDa protein
 JUND Transcription factor jun-D
 MARS Methionyl-tRNA synthetase, cytoplasmic
 TAGLN3 Transgelin-3
 PPP5C Serine/threonine-protein phosphatase 5
 NCAM1 Isoform 3 of Neural cell adhesion molecule 1
 MESDC2 Mesoderm development candidate 2
 SF3A2 SF3A2 protein (Fragment)
 CHCHD2 Coiled-coil-helix-coiled-coil-helix domain-containing protein 2, mitochondrial
 ANKRD30A Ankyrin repeat domain-containing protein 30A
 RBBP7 Histone-binding protein RBBP7
 ECHS1 Enoyl-CoA hydratase, mitochondrial
 NUDT21 Cleavage and polyadenylation specificity factor subunit 5
 TXLNA Alpha-taxilin
 CCDC47 Isoform 1 of Coiled-coil domain-containing protein 47
 ARPC5 Isoform 1 of Actin-related protein 2/3 complex subunit 5
 SRP72 CaM kinase II isoform
 CSNK2A1P;CSNK2A1 Casein kinase II alpha subunit
 CHD4 Isoform 1 of Chromodomain-helicase-DNA-binding protein 4
 SNRPC U1 small nuclear ribonucleoprotein C
 HNRPDL Isoform 1 of Heterogeneous nuclear ribonucleoprotein D-like
 CCBL2 kynurenine aminotransferase III isoform 3
 LUC7L2 Isoform 1 of Putative RNA-binding protein Luc7-like 2
 MRPL45 39S ribosomal protein L45, mitochondrial
 GTF2F2 General transcription factor IIF subunit 2
 C22orf28 UPF0027 protein C22orf28
 NEDD8 NEDD8
 ZNF781 Isoform 1 of Zinc finger protein 781
 CDIPT Isoform 1 of CDP-diacylglycerol--inositol 3-phosphatidyltransferase
 ANXA5 Annexin A5
 PTGES3 Prostaglandin E synthase 3
 CHCHD3 Coiled-coil-helix-coiled-coil-helix domain-containing protein 3, mitochondrial
 EFTUD2 116 kDa U5 small nuclear ribonucleoprotein component
 TERF2IP Telomeric repeat-binding factor 2-interacting protein 1
 CSDE1 Isoform Long of Cold shock domain-containing protein E1
 DDX23 Probable ATP-dependent RNA helicase DDX23
 NAPA Alpha-soluble NSF attachment protein
 C1orf61 Protein
 MAP7D2 Isoform 2 of MAP7 domain-containing protein 2
 LOC100130999;FAM81A hypothetical protein LOC145773
 TMSB10 Thymosin beta-10
 LOC731605 hypothetical LOC731605
 RBM14 Isoform 1 of RNA-binding protein 14
 RPL13A 60S ribosomal protein L13a
 CAMKK2 Isoform 3 of Calcium/calmodulin-dependent protein kinase kinase 2
 ANP32E Acidic leucine-rich nuclear phosphoprotein 32 family member E
 RPA1 Replication protein A 70 kDa DNA-binding subunit
 PSMA4 Proteasome subunit alpha type-4
 LOC51035 Isoform 1 of SAPK substrate protein 1
 ZSWIM6 zinc finger, SWIM domain containing 6
 SMC2 Isoform 1 of Structural maintenance of chromosomes protein 2
 RORB Isoform 2 of Nuclear receptor ROR-beta
 UFD1L Isoform Short of Ubiquitin fusion degradation protein 1 homolog
 WIZ Isoform 2 of Protein Wiz
 C10orf58 Uncharacterized protein C10orf58
 SYN2 Uncharacterized protein SYN2 (Fragment)
 DDR2 Discoidin domain receptor tyrosine kinase 2
 CAD CAD protein
 EIF4A3 Eukaryotic initiation factor 4A-III
  Protein Mascot Score  # of peptides  log2(heavy /light)  58
 76
 27
 152
 47
 50
 27
 525
 110
 55
 225
 102
 66
 184
 184
 63
 29
 32
 118
 95
 32
 48
 76
 26
 129
 61
 228
 65
 48
 160
 477
 54
 45
 53
 82
 25
 166
 49
 68
 35
 214
 130
 101
 28
 76
 25
 37
 60
 27
 26
 29
 32
 247
 144
 42
 63
 120
 33
 36
 36
 40
 29
 54
 29
 152
 35
 34
 698
 95
 61
 33
 26
 139
 26
 79
 48
 41
 65
 27
 626
 34
  2
 2
 1
 2
 1
 1
 1
 7
 2
 2
 4
 3
 2
 5
 3
 1
 1
 1
 2
 3
 1
 1
 2
 1
 3
 1
 2
 1
 1
 3
 7
 2
 1
 1
 1
 1
 3
 1
 2
 1
 6
 3
 3
 1
 2
 1
 1
 2
 1
 1
 1
 1
 6
 3
 1
 2
 2
 1
 1
 1
 1
 1
 1
 1
 3
 1
 1
 8
 2
 1
 1
 1
 3
 1
 2
 1
 1
 2
 1
 14
 1
  -0.78
 -0.78
 -0.78
 -0.79
 -0.79
 -0.80
 -0.80
 -0.81
 -0.82
 -0.82
 -0.82
 -0.82
 -0.83
 -0.83
 -0.83
 -0.83
 -0.83
 -0.83
 -0.84
 -0.84
 -0.84
 -0.85
 -0.85
 -0.86
 -0.86
 -0.87
 -0.87
 -0.87
 -0.87
 -0.88
 -0.88
 -0.88
 -0.89
 -0.89
 -0.90
 -0.90
 -0.90
 -0.90
 -0.90
 -0.90
 -0.90
 -0.91
 -0.91
 -0.91
 -0.94
 -0.95
 -0.95
 -0.96
 -0.96
 -0.97
 -0.97
 -0.97
 -0.98
 -0.99
 -0.99
 -1.00
 -1.00
 -1.00
 -1.01
 -1.02
 -1.02
 -1.03
 -1.04
 -1.04
 -1.04
 -1.04
 -1.05
 -1.05
 -1.06
 -1.06
 -1.07
 -1.07
 -1.07
 -1.07
 -1.08
 -1.10
 -1.10
 -1.11
 -1.11
 -1.12
 -1.13
  110  Proteins eluted with mild detergent and salt (weak parkin interactors – CCCP conditions) ARGLU1 Isoform 1 of Arginine and glutamate-rich protein 1
 FKSG30 Kappa-actin
 PELO Protein pelota homolog
 KIF15 Isoform 4 of Kinesin-like protein KIF15
 GLS Isoform GAC of Glutaminase kidney isoform, mitochondrial
 NPLOC4 Isoform 2 of Nuclear protein localization protein 4 homolog
 LSM4 U6 snRNA-associated Sm-like protein LSm4
 EIF5 Eukaryotic translation initiation factor 5
 VPS13C Isoform 1 of Vacuolar protein sorting-associated protein 13C
 DDC Aromatic-L-amino-acid decarboxylase
 MTIF3 Translation initiation factor IF-3, mitochondrial
 CDC73 Putative uncharacterized protein (Fragment)
 RANBP3 Isoform 1 of Ran-binding protein 3
 - NEFM protein
 BAT3 Isoform 1 of Large proline-rich protein BAT3
 IGF2BP3 Isoform 1 of Insulin-like growth factor 2 mRNA-binding protein 3
 CCDC49 24 kDa protein
 LUC7L Isoform 1 of Putative RNA-binding protein Luc7-like 1
 DOCK1 Dedicator of cytokinesis protein 1
 VIM Vimentin
 THRAP3 Thyroid hormone receptor-associated protein 3
 TPT1 Tumor protein, translationally-controlled 1
 HDAC2 histone deacetylase 2
 DLG1 Isoform 2 of Disks large homolog 1
 DCTN1 Isoform p150 of Dynactin subunit 1
 INA Alpha-internexin
 HNRNPH3 Isoform 1 of Heterogeneous nuclear ribonucleoprotein H3
 - 71 kDa protein
 - SMT3 suppressor of mif two 3 homolog 2 isoform b precursor
 PHOX2A Paired mesoderm homeobox protein 2A
 TERF2 Isoform 1 of Telomeric repeat-binding factor 2
 HNRNPC Isoform C1 of Heterogeneous nuclear ribonucleoproteins C1/C2
 - Pseudogene candidate
 PRPH Isoform 1 of Peripherin
 YWHAH 14-3-3 protein eta
 ADAR Isoform 2 of Double-stranded RNA-specific adenosine deaminase
 ZNF415 Isoform 5 of Zinc finger protein 415
 STX5 Isoform 2 of Syntaxin-5
 SYNE2 Isoform 1 of Nesprin-2
 NHSL2 NHS-like protein 2
 ALDH2 Aldehyde dehydrogenase, mitochondrial
 ACTR3 Actin-related protein 3
 RAB2B cDNA FLJ14824 fis, clone OVARC1000771, moderately similar to RAS-RELATED PROTEIN RAB-2
 PHTF1 Putative homeodomain transcription factor 1
 - 3 kDa protein
 FLNB Isoform 1 of Filamin-B
 SYT1 Synaptotagmin-1
 AP3M1 AP-3 complex subunit mu-1
 KIF15 Isoform 1 of Kinesin-like protein KIF15
 RDX Radixin isoform b
 ATP5H Isoform 1 of ATP synthase subunit d, mitochondrial
 SLC38A8 Putative sodium-coupled neutral amino acid transporter 8
 LEMD3 Inner nuclear membrane protein Man1
 PABPN1 Isoform 1 of Polyadenylate-binding protein 2
 RBM39 Isoform 1 of RNA-binding protein 39
 ZSCAN21 Zinc finger and SCAN domain-containing protein 21
 CORO7;Magmas Coronin-7
 APAF1 Isoform 1 of Apoptotic protease-activating factor 1
 NEFL Putative uncharacterized protein DKFZp761K0922 (Fragment)
 C1orf87 Isoform 1 of Uncharacterized protein C1orf87
 SOX13 SRY-box 13
 RAB40A Ras-related protein Rab-40A
 FAM13A1 family with sequence similarity 13, member A1 isoform a
 ADCK1 Isoform 2 of Uncharacterized aarF domain-containing protein kinase 1
 CD320 CD320 antigen
 MAST2 MAST2 protein
 C1orf168 Isoform 1 of Uncharacterized protein C1orf168
 S100A7 Protein S100-A7
 HNRNPA0 Heterogeneous nuclear ribonucleoprotein A0
 - 12 kDa protein
 BEST1 Isoform 1 of Bestrophin-1
 PARK2 Isoform 1 of E3 ubiquitin-protein ligase parkin
 STX18 Syntaxin-18
 STRA8 Stimulated by retinoic acid gene 8 protein homolog
 KIAA0564 Isoform 3 of Uncharacterized protein KIAA0564
 AGRN Agrin
 PDE4C PDE4C-delta109
 LOC646821 similar to beta-actin
 RAB8B 10 kDa protein
 CCDC66 51 kDa protein
  Protein Mascot Score  # of peptides  log2(heavy /light)  29
 28
 26
 25
 185
 63
 33
 71
 64
 78
 30
 29
 188
 357
 73
 92
 33
 171
 25
 1015
 213
 31
 166
 57
 35
 620
 254
 64
 88
 26
 35
 164
 53
 475
 87
 69
 62
 34
 26
 54
 28
 50
  1
 1
 1
 1
 3
 2
 1
 2
 2
 2
 1
 1
 3
 5
 2
 1
 1
 4
 1
 19
 5
 1
 4
 2
 1
 11
 4
 2
 1
 1
 1
 4
 1
 9
 1
 1
 2
 1
 1
 1
 1
 1
  -1.13
 -1.13
 -1.13
 -1.13
 -1.14
 -1.14
 -1.15
 -1.15
 -1.16
 -1.16
 -1.16
 -1.18
 -1.19
 -1.21
 -1.24
 -1.26
 -1.27
 -1.28
 -1.28
 -1.29
 -1.29
 -1.29
 -1.30
 -1.32
 -1.32
 -1.33
 -1.33
 -1.37
 -1.41
 -1.41
 -1.44
 -1.44
 -1.51
 -1.52
 -1.52
 -1.53
 -1.55
 -1.63
 -1.67
 -1.68
 -1.68
 -1.78
  29
 28
 25
 45
 55
 26
 30
 49
 33
 27
 32
 27
 26
 25
 178
 37
 139
 26
 59
 30
 26
 33
 30
 25
 27
 56
 32
 25
 32
 41
 26
 35
 28
 28
 26
 96
 26
 56
  1
 1
 1
 1
 1
 1
 1
 1
 1
 1
 1
 1
 1
 1
 4
 1
 3
 1
 2
 1
 1
 1
 1
 1
 1
 1
 1
 1
 1
 1
 1
 1
 1
 1
 1
 3
 1
 2
  -1.78
 -1.78
 -1.79
 -1.80
 -1.85
 -1.89
 -1.94
 -2.01
 -2.02
 -2.06
 -2.08
 -2.09
 -2.20
 -2.30
 -2.31
 -2.31
 -2.35
 -2.51
 -2.52
 -2.53
 -2.56
 -2.58
 -2.59
 -2.61
 -2.67
 -2.71
 -2.75
 -2.87
 -2.89
 -2.91
 -2.94
 -3.50
 -3.52
 -3.52
 -3.79
 -3.84
 -3.96
 -4.12
  111  Proteins eluted with mild detergent and salt (weak parkin interactors – CCCP conditions) DUSP19 Isoform 1 of Dual specificity protein phosphatase 19
 VWA3A cDNA FLJ40941 fis, clone UTERU2008426
 MOS Proto-oncogene serine/threonine-protein kinase mos
 - Ig kappa chain V-III region GOL
 SLC12A2 Isoform 1 of Solute carrier family 12 member 2
 SPATA5 Isoform 1 of Spermatogenesis-associated protein 5
 CEACAM18 Carcinoembryonic antigen-related cell adhesion molecule 18
 STAB1 Isoform 2 of Stabilin-1
 LOC389765 Uncharacterized protein ENSP00000297820
 PPM1H Protein phosphatase 1H
 - Hypothetical short protein
 RALY Putative uncharacterized protein (Fragment)
 LGI3 Leucine-rich repeat LGI family member 3
 LOC121006 hypothetical protein
 CBFA2T3 Isoform 2 of Protein CBFA2T3
 NRG1 Isoform 3 of Pro-neuregulin-1, membrane-bound isoform
  Protein Mascot Score  # of peptides  log2(heavy /light)  26
 29
 26
 29
 37
 50
 27
 29
 52
 70
 26
 86
 28
 28
 26
 52
  1
 1
 1
 1
 1
 2
 1
 1
 2
 2
 1
 2
 1
 1
 1
 2
  -4.15
 -4.26
 -4.26
 -4.33
 -4.64
 -4.64
 -4.79
 -5.15
 -5.46
 -5.46
 -5.54
 -5.84
 -6.08
 -6.22
 -6.74
 -7.54
  112  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.24.1-0068961/manifest

Comment

Related Items