UBC Faculty Research and Publications

SNAP-25 is a promising novel cerebrospinal fluid biomarker for synapse degeneration in Alzheimer’s disease Brinkmalm, Ann; Brinkmalm, Gunnar; Honer, William G; Frölich, Lutz; Hausner, Lucrezia; Minthon, Lennart; Hansson, Oskar; Wallin, Anders; Zetterberg, Henrik; Blennow, Kaj; Öhrfelt, Annika Nov 23, 2014

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RESEARCH ARTICLESNAP-25 is a promising nogLuj Bhave directly demonstrated loss of presynaptic proteinsand synaptic dysfunction [1,2]. In patients, however,system synaptosomal-associated protein 25 (SNAP-25) isan important marker of functional synapses, being oneBrinkmalm et al. Molecular Neurodegeneration 2014, 9:53http://www.molecularneurodegeneration.com/content/9/1/53only trace amounts, and the membrane-bound nature ofS-431 80 Mölndal, SwedenFull list of author information is available at the end of the articleassays for presynaptic proteins are indirect or rely onpost-mortem findings. In the early stages of disease theyhave provided inconsistent results reporting elevated, un-changed, and lower protein amounts [3-12]. Biomarkerstudies of amyloidβ1-42 (Aβ1-42), total tau (T-tau) andtau phosphorylated at threonine 181 (P-tau181) in cerebro-spinal fluid (CSF) have contributed to understanding theessential component of the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE)complex. These proteins mediate synaptic communicationby initiating fusion of synaptic vesicles [14].The notions that synaptic loss occurs early in Alzheimer’sdisease, and that synaptic proteins at active synapses couldbe biomarkers indicating the degree of synaptic degene-ration have prompted interest in detecting relevant synap-tic proteins in human biological fluid samples. Analysis ofsynaptic proteins in CSF is complicated by the presence of* Correspondence: ann.brinkmalm@neuro.gu.se1Institute of Neuroscience and Physiology, Department of Psychiatry andNeurochemistry, Sahlgrenska Academy at the University of Gothenburg,Background: Synaptic degeneration is an early pathogenic event in Alzheimer’s disease, associated with cognitiveimpairment and disease progression. Cerebrospinal fluid biomarkers reflecting synaptic integrity would be highlyvaluable tools to monitor synaptic degeneration directly in patients. We previously showed that synaptic proteinssuch as synaptotagmin and synaptosomal-associated protein 25 (SNAP-25) could be detected in pooled samples ofcerebrospinal fluid, however these assays were not sensitive enough for individual samples.Results: We report a new strategy to study synaptic pathology by using affinity purification and mass spectrometryto measure the levels of the presynaptic protein SNAP-25 in cerebrospinal fluid. By applying this novel affinity massspectrometry strategy on three separate cohorts of patients, the value of SNAP-25 as a cerebrospinal fluid biomarkerfor synaptic integrity in Alzheimer’s disease was assessed for the first time. We found significantly higher levels ofcerebrospinal fluid SNAP-25 fragments in Alzheimer’s disease, even in the very early stages, in three separatecohorts. Cerebrospinal fluid SNAP-25 differentiated Alzheimer’s disease from controls with area under the curve of0.901 (P < 0.0001).Conclusions: We developed a sensitive method to analyze SNAP-25 levels in individual CSF samples that to ourknowledge was not possible previously. Our results support the notion that synaptic biomarkers may be importanttools for early diagnosis, assessment of disease progression, and to monitor drug effects in treatment trials.Keywords: Alzheimer’s disease, Biomarker, Cerebrospinal fluid, SNAP-25, SNARE proteins, Mass spectrometry,Immunopurification, Selected reaction monitoringBackgroundAnimal models of the early phases of Alzheimer’s diseasesequence of clinically relevant molecular events contribut-ing to cognitive impairment [13]. In the central nervousbiomarker for synapse deAlzheimer’s diseaseAnn Brinkmalm1*, Gunnar Brinkmalm1, William G Honer2,Oskar Hansson4,5, Anders Wallin1, Henrik Zetterberg1,6, KaAbstract© 2014 Brinkmalm et al.; licensee BioMed CenCreative Commons Attribution License (http:/distribution, and reproduction in any mediumDomain Dedication waiver (http://creativecomarticle, unless otherwise stated.Open Accessvel cerebrospinal fluideneration intz Frölich3, Lucrezia Hausner3, Lennart Minthon4,5,lennow1 and Annika Öhrfelt1tral Ltd. This is an Open Access article distributed under the terms of the/creativecommons.org/licenses/by/4.0), which permits unrestricted use,, provided the original work is properly credited. The Creative Commons Publicmons.org/publicdomain/zero/1.0/) applies to the data made available in thismany of these proteins [15]. Several research groups, in-cluding our own, have detected synaptic proteins in CSF[16-21]. However, these studies were performed on rela-tively large quantities of pooled CSF from multiple pa-tients [16,17,21]. Moreover, the target proteins had to beselectively purified and concentrated in several steps andthe quantitative aspects of the techniques may have beensub-optimal [20].Here, we have developed an assay where the concen-tration of the presynaptic protein SNAP-25 could bereproducibly measured in CSF samples from individualpurifying with SMI81 instead of SP12, SNAP-25 could bequantified in all soluble fractions (Figure 1C). Never-theless, no significant differences between the levels ofsoluble SNAP-25 in Alzheimer’s disease and control brainhomogenate samples were observed.To investigate if the dissimilar levels of SP12- andSMI81-immunoreactive SNAP-25 in the soluble fractionscould be due to a truncation or other post-translationalmodifications we analyzed a subset of the samples withLC-MS/MS. In the majority of the soluble fractions, onlytryptic peptides originating from the N-terminal part ofdivndariBrinkmalm et al. Molecular Neurodegeneration 2014, 9:53 Page 2 of 13http://www.molecularneurodegeneration.com/content/9/1/53patients. We hypothesized that soluble forms of brainSNAP-25 were the most likely to resemble SNAP-25 inCSF. Since SNAP-25 is abundant in brain tissue we usedbiochemically fractionated human brain homogenate(soluble, membrane-bound, and membrane-raft asso-ciated protein fractions) to design a strategy for quan-tification of SNAP-25 in CSF by combining selectivepurification with immunoprecipitation, digestion withtrypsin, and mass spectrometry analysis. We found sig-nificantly higher levels of SNAP-25 in CSF in Alzheimer’sdisease in three separate cohorts, including in the veryearly stage of the disease.ResultsCharacterization and quantification of SNAP-25 in humanbrain tissueWe used the monoclonal antibodies to affinity purifySNAP-25 from biochemically fractionated human brainhomogenate. Using the SP12 antibody and quantificationwith selected reaction monitoring mass spectrometry(SRM-MS) we compared the SNAP-25 levels in brainhomogenate fractions from Alzheimer’s disease patients(N = 15) and age-matched controls (N = 15) (Additionalfile 1: Table S1). We found that the levels of SNAP-25were significantly lower in the Alzheimer’s disease groupfor the membrane-bound and the membrane-raft asso-ciated fractions (Figure 1A-B). In contrast, the levels ofSNAP-25 in the soluble protein fractions were very low orundetectable (data not shown). However, when affinityFigure 1 Targeted SRM-MS analyses of SNAP-25 in human brain. Inlabeled peptide standard) of immunoprecipitated [antibodies SP12 (A-B) a(A) and membrane-raft associated (B) and soluble (C), extract of superior p(N = 15). The lower, upper and middle lines of the error bars correspond to ththe extreme N-terminus of SNAP-25, especially when it’s N-terminal acetylatedthe SNAP-25 protein were detected (Figure 2), indicatingthat the soluble SNAP-25 were C-terminally truncated.However, in all the membrane-bound and membrane-raftassociated fractions the entire SNAP-25 protein wasdetected (regardless of antibody) (Additional file 1:Figure S1). Moreover, in all fractions, including thesoluble, SNAP-25 was N-terminally modified by me-thionine excision and acetylation.To characterize the soluble SNAP-25 forms we used atop-down LC-MS/MS approach. Using undigested pro-teins from the SMI81 affinity purified soluble fraction, wesuccessfully identified eight truncated forms of solubleSNAP-25, all N-terminally modified by methionine exci-sion and acetylation (Additional file 1: Figure S2, TableS2). Building on the findings of the soluble truncatedSNAP-25 peptides we changed our approach to targetthe furthermost N-terminal tryptic peptides of SNAP-25(Figure 2B) and perform the MS-based quantification withhigh resolution selected ion monitoring (HR-SIM-MS) ona Quadrupole-Orbitrap Mass Spectrometer (Q Exactive)[22]. The reproducibility of the novel method was mea-sured and the CV of the SNAP-25 levels was found to beless than 10% (Additional file 1: Table S3).Evaluation of SNAP-25 as a synaptic marker in CSFsamplesDemographic resultsTable 1 shows the demographic characteristics of thegroups. The German cohort was composed of nine patientsidual values for the SRM-MS measured ratios (endogenous peptide/SMI81 (C) SNAP-25, in biochemically fractionated membrane-boundetal gyrus from controls (N = 15) and patients with Alzheimer’s diseasee 25th and 75th percentiles and medians, respectively. SMI81 recognizes. The exact epitope of SP12 is unknown.Brinkmalm et al. Molecular Neurodegeneration 2014, 9:53 Page 3 of 13http://www.molecularneurodegeneration.com/content/9/1/53with Alzheimer’s disease (three men and six women, 62-83years), seven subjects with prodromal Alzheimer’s disease(four men and three women, 57-77 years), and nine non-demented controls (two men and seven women, 60-83years). The first replication set (Swedish cohort I) was com-posed of 10 patients with Alzheimer’s disease (three menand seven women, 59-84 years), and six non-dementedcontrols (one man and five women, 47-64 years). The sec-ond replication set (Swedish cohort II) was composed of 17patients with Alzheimer’s disease (five men and 12 women,61-76 years), and 17 healthy controls (nine men and eightwomen, 63-70 years). The patients and controls in theGerman and Swedish cohort II were age-matched, whilethe patients with Alzheimer’s disease were significantlyFigure 2 Sequence coverage of SNAP-25. (A) Heat map for the relativeBiochemically fractionated soluble proteins of superior parietal gyrus fromimmunoprecipitated with SMI81. High resolution LC-MS ion chromatogramAn increase of signal intensity is seen as a blue to red shift. (B) Amino acididentified by top-down LC-MS/MS analysis are indicated by arrows above tindicated by boxes. Amino acids belonging to tryptic peptides identified b0 layer” contribute together with a syntaxin glutamine (Q), and a VAMP argSNARE complex.older than the controls in the Swedish cohort I. In all threecohorts, patients with Alzheimer’s disease had a signifi-cantly lower MMSE score compared with the controls.Levels of SNAP-25 in CSFThe CSF levels of all three investigated tryptic peptides ofSNAP-25 were significantly higher in patients with pro-dromal Alzheimer’s disease and overt Alzheimer’s diseasecompared with non-demented controls (Figure 3A-C).Moreover, two of the SNAP-25 peptides (amino acids17-31 and 32-40) were significantly higher in Alzheimer’sdisease compared with prodromal Alzheimer’s disease(Figure 3B-C). Consistently, the CSF levels of all trypticpeptides of SNAP-25 were significantly higher in patientsLC-MS signal intensities of individual tryptic peptides from SNAP-25B.controls (N = 7) and patients with Alzheimer’s disease (N = 9) weres of all peptides identified as belonging to SNAP-25B were extracted.sequences for SNAP-25B and SNAP-25A. Soluble endogenous formshe sequences. Differences between SNAP-25B and SNAP-25A arey LC-MS/MS are labeled bold. The two glutamines (Q) indicated “ionicinine (R) to the highly conserved 3Q:1R motif at the core of thee dal))-75-51) PalalBrinkmalm et al. Molecular Neurodegeneration 2014, 9:53 Page 4 of 13http://www.molecularneurodegeneration.com/content/9/1/53Table 1 Demographic data and biomarker CSF levels for thGerman cohort Control ProdromNumber (Men/Women) 9 (2/7) 7 (4/3)Age (years) 70 (68-74) 72 (69-73MMSE 27 (25-29) 28 (27-28Aβ1-42 (ng/L) 1065 (797-1201) 541 (521T-tau (ng/L) 165 (135-208) 403 (353P-tau181 (ng/L) 45 (38-50) 81 (76-95Swedish cohort I Control ProdromNumber (Men/Women) 6 (1/5)Age (years) 54 (48-63)MMSE 27 (27-28)Aβ1-42 (ng/L) 915 (860-1040)T-tau (ng/L) 290 (230-300)P-tau181 (ng/L) 56 (40-60)Swedish cohort II Control ProdromNumber (Men/Women) 17 (9/8)Age (years) 66 (64-68)MMSE 30 (29-30)with Alzheimer’s disease compared with non-dementedcontrols in the replication set (Figure 3D-F). In the secondreplication set, the CSF levels of the tryptic peptide ofSNAP-25 (32-40) were significantly higher in patientswith Alzheimer’s disease compared with healthy controls(Figure 3I).There were no cohort effects on the CSF levels ofnovel SNAP-25 biomarkers, P-tau181 or Aβ1-42 (datanot shown), allowing statistical analyses of the entiregroup of participant samples from Alzheimer’s disease(N = 36) and controls (N = 32) (Figure 3J-L). The trypticpeptide assays of SNAP-25 (32-40, 17-31, and Ac-2-16)and CSF biomarkers (Aβ1-42, T-tau and P-tau181) couldeach differentiate Alzheimer’s disease (N = 36) from con-trols (N = 32), with area under the curve of 0.901 (0.828-0.974) (P < 0.0001), 0.808 (0.703-0.913) (P < 0.0001), 0.772(0.659-0.885) (P < 0.001), 0.881 (0.802-0.960) (P < 0.0001),0.933 (0.873-0.994) (P < 0.0001), and 0.916 (0.844-0.987)(P < 0.0001), respectively (Figure 4).No statistically significant correlations between ageand levels of the tryptic peptides of SNAP-25 (Ac-2-16,17-31, and 32-40) were observed in either the controlgroup (N = 33) or the Alzheimer’s disease group (N = 36)(Table 2). The MMSE score, indicating the severity ofAβ1-42 (ng/L) 640 (530-870)T-tau (ng/L) 250 (180-320)P-tau181 (ng/L) 46 (34-58)Abbreviations: Aβ1-42 (amyloidβ 1-42), CSF (cerebrospinal fluid), MMSE (mini-mentathreonine 181).aData are given as median (interquartile range) unless otherwise indicated. StatisticbCompared with controls.cCompared with prodromal Alzheimer’s disease.iagnostic groupsaAlzheimer’s disesase Alzheimer’s disease9 (3/6)68 (68-79)22 (21-23) P = 0.02b, P = 0.001c3) P = 0.005b 524 (424-695) P = 0.0005b3) P = 0.0002b 779 (683-864) P = 0.00004b, P = 0.002c= 0.0002b 130 (108-161) P = 0.00004b, P = 0.005cAlzheimer’s disesase Alzheimer’s disease10 (3/7)77 (73-82) P = 0.001b24 (22-25) P = 0.0003b470 (355-560) P = 0.0003b690 (590-1100) P = 0.0002b92 (84-132) P = 0.0003bAlzheimer’s disesase Alzheimer’s disease17 (5/12)68 (66-70)25 (24-27) P < 0.0001bcognitive impairment, correlated significantly with SNAP-25(Ac-2-16) in the Alzheimer’s disease (N = 36) group(Table 2). All tryptic peptides of SNAP-25 (Ac-2-16,17-31, and 32-40) correlated with the levels of T-tau andP-tau181 in both the control group (N = 33) and in patientswith Alzheimer’s disease (N = 36) (Table 2). The trypticpeptides of SNAP-25 (Ac-2-16 and 17-31) correlated withAβ1-42 in the control group, but not in patients withAlzheimer’s disease (Table 2).DiscussionWe report a new strategy to study synaptic pathology byusing affinity purification and quantitative mass spec-trometry to measure levels of the presynaptic SNAP-25in CSF samples from individual patients. This is the firststudy demonstrating that SNAP-25 might be a usefulCSF biomarker in differential diagnosis of patients withAlzheimer’s disease/prodromal Alzheimer’s disease fromcontrols and also in discriminating Alzheimer’s diseasefrom prodromal Alzheimer’s disease.The novel CSF SNAP-25 assay was developed usingbiochemically fractionated human brain homogenatewith high concentration of synaptic proteins. We foundthat the levels of SNAP-25 were significantly lower in320 (210-440) P = 0.0001b560 (360-1020) P = 0.0006b93 (54-127) P = 0.001bl state examination), T-tau (total tau), P-tau181 (tau phosphorylated atal differences were determined using nonparametric tests.Figure 3 (See legend on next page.)Brinkmalm et al. Molecular Neurodegeneration 2014, 9:53 Page 5 of 13http://www.molecularneurodegeneration.com/content/9/1/53the Alzheimer’s disease group for the membrane-bound andthe membrane-raft associated fractions. These results areconsistent with previous studies of SNAP-25 in Alzheimer’sdisease using different immunoassays [5,6,8,23,24] anddemonstrated that the method was sufficiently sensitive todetect pathological changes in brain tissue samples fromindividual patients.The dissimilar levels of SP12- and SMI81-immunoreactiveSNAP-25 in the soluble fractions made us hypothesizethat soluble SNAP-25 may be truncated or modified com-pared to membrane associated SNAP-25. We found thatonly tryptic peptides originating from the N-terminal partof the soluble SNAP-25 protein were detected. Moreover,in all fractions, including the soluble, SNAP-25 wasN-terminally modified by methionine excision and acetyl-ation, consistent with a previous study [25]. The existenceof a soluble N-terminal peptide fragment of SNAP-25 isconsistent with the observations that immunoprecipi-tation with a monoclonal antibody directed towards the(See figure on previous page.)Figure 3 Targeted HR-SIM-MS analyses of SNAP-25 in human CSF. Indpeptide/labeled peptide standard] multiplied by 10,000) of immunoprecipitat(A-C), Swedish cohort I (D-F), Swedish cohort II (G-I), and the entire group ofdepict the measured levels of three N-terminal tryptic peptides of SNAP-25, ABrinkmalm et al. Molecular Neurodegeneration 2014, 9:53 Page 6 of 13http://www.molecularneurodegeneration.com/content/9/1/53N-terminal of SNAP-25 increases the yield.Figure 4 ROC curve analysis of SNAP-25 in human CSF. ROCcurve analysis for SNAP-25 32-40 (turquoise), SNAP-25 17-31 (green),SNAP-25 Ac-2-16 (black) Aβ1-42 (orange), T-tau (purple) and P-tau181(yellow) in CSF for differentiation of Alzheimer’s disease patientsfrom controls in the entire subject material. The area under thecurve (95% confidence interval) was 0.901 (0.828-0.974) (P < 0.0001),0.808 (0.703-0.913) (P < 0.0001), 0.772 (0.659-0.885) (P < 0.001), 0.881(0.802-0.960) (P < 0.0001), 0.933 (0.873-0.994) (P < 0.0001), and 0.916(0.844-0.987) (P < 0.0001), respectively.Truncated soluble forms of SNAP-25 had never beenreported before and we characterized the various formswith a top-down tandem MS approach. Undigested pro-teins from the SMI81 affinity purified soluble fractionwere analyzed directly on LC-MS/MS. We successfullyidentified eight truncated forms of soluble SNAP-25, allN-terminally modified by methionine excision and acety-lation. An interesting finding was that the potential clea-vage site for the creation of the longest truncated form ofSNAP-25 (Ac-2-47) is located very close to the ioniczero layer at the center of the SNARE complex [26,27](Figure 2B). However, the longest soluble SNAP-25 con-tains the N-terminal amino acids 2-47 of the protein whilethe two isoforms SNAP-25A and SNAP-25B differs inamino acids 58, 60, 65, 69, 79, 84, and 88-89. Hence,soluble SNAP-25 no longer contains information regar-ding its original isoform (Figure 2B).We found that the truncated forms of SNAP-25 werepresent in CSF, and the level of the tryptic peptide ofSNAP-25 (32-40) was consistently and significantly higherin patients with Alzheimer’s disease compared with con-trols in the three independent cohorts. The tryptic peptideassays of SNAP-25 (32-40, 17-31, and Ac-2-16) could eachdifferentiate Alzheimer’s disease from controls. However,the tryptic peptide assay of SNAP-25 (32-40) provided aslightly better differentiation of patients with Alzheimer’sdisease from controls compared with the tryptic assays ofSNAP-25 (Ac-2-16 and 17-31). The CSF levels of two ofthe tryptic peptides of SNAP-25 (Ac-2-16 and 17-31) weresignificantly higher in patients with Alzheimer’s diseasecompared with controls in two of the three examinedclinical cohorts. Summarizing, these findings suggestSNAP-25 (32-40) to provide the best differential diagnos-tic biomarker of Alzheimer’s disease and showed differen-tiation of patients with Alzheimer’s disease from controlsin a similar magnitude as the CSF biomarkers (Aβ1-42,T-tau and P-tau181).In the early stages of disease, synaptic markers in pre-vious studies provided inconsistent results reporting ele-vated, unchanged, and lower protein amounts [3-12]. Inividual values for the HR-SIM-MS measured peak area ratios ([endogenoused (SMI81 antibody) SNAP-25 in CSF samples within German cohortAlzheimer’s disease (N = 36) and controls (N = 32) (J-L). The nine panelsc-2-16 (A, D, G, J), 17-31 (B, E, H, K), and 32-40 (C, F, I, L).the present study, CSF SNAP-25 peptides were alreadyincreased in prodromal Alzheimer’s disease comparedwith controls, supporting the notion that this synapticmarker might provide an early marker for Alzheimer’s dis-ease. Two of the SNAP-25 peptides (17-31 and 32-40)could also be used to differentiate prodromal Alzheimer’sdisease and overt Alzheimer’s disease. A limitation of thisstudy is the small sample size for patients with mildelalsigBrinkmalm et al. Molecular Neurodegeneration 2014, 9:53 Page 7 of 13http://www.molecularneurodegeneration.com/content/9/1/53cognitive impairment, therefore the value of SNAP-25 asan early biomarker remains to be established.The patients and controls in two of the cohorts wereage-matched, while the patients with Alzheimer’s diseaseTable 2 Correlation between age, MMSE and biomarker levSNAP-25, Ac-2-16Cont (N = 33)Age N.S.MMSE N.S.Aβ1-42 rho = 0.560, P = 0.001T-tau rho = 0.564, P = 0.001P-tau181 rho = 0.735, P < 0.0001SNAP-25, Ac-2-16 -SNAP-25, 17-31 -AD (N = 36)Age N.S.MMSE rho = -0.509, P = 0.002Aβ1-42 N.S.T-tau rho = 0.557, P = 0.0004P-tau181 rho = 0.717, P < 0.0001SNAP-25, Ac-2-16 -SNAP-25, 17-31 -Abbreviations: Aβ1-42 (amyloidβ 1-42), AD (Alzheimer’s disease), CSF (cerebrospin(tau phosphorylated at threonine 181).aCorrelations presented by the Spearman’s rank correlation coefficient (rho). Nonwere significantly older than the controls in the Swedishcohort I. However, no statistically significant correlationsbetween age and levels of the tryptic peptides of SNAP-25(Ac-2-16, 17-31, and 32-40) in either the control group orthe Alzheimer’s disease group were observed, suggestingthat the detected SNAP-25 fragments not are influencedby age.To date there is no CSF biomarker available thatmakes it possible to follow the progression of cognitivedecline. Previous studies suggest that synaptic loss corre-lates with the clinical manifestations of Alzheimer’s dis-ease, while there is no relation between the number ofaccumulated parenchymal amyloid plaques and synapticpathology [3,4]. In the present study, there was a negativecorrelation between SNAP-25 (Ac-2-16) and the MMSE,indicating patients suffering from more severe cognitivedecline had higher levels of SNAP-25 (Ac-2-16), whichimplies that the novel biomarker might be useful to followprogression of cognitive decline. Interestingly, previousstudies have shown evidence that SNAP-25 single nu-cleotide polymorphisms are associated with cognitivedecline [28,29].The CSF level of T-tau generally reflects the intensity ofaxonal and neuronal degeneration occurring in brain, whileP-tau181 serves as a more specific marker for Alzheimer’sdisease [30] CSF T-tau, P-tau181 and Aβ1-42 are stableover time making these Alzheimer’s biomarkers feasiblefor monitoring biochemical effects in clinical trials [31].The finding that all investigated SNAP-25 peptides corre-lated well with T-tau and P-tau181, suggests that SNAP-25s in the entire CSF materialaSNAP-25, 17-31 SNAP-25, 32-40N.S. N.S.N.S. N.S.rho = 0.468, P = 0.007 N.S.rho = 0.563, P = 0.001 rho = 0.656, P = 0.0001rho = 0.715, P < 0.0001 rho = 0.659, P < 0.0001rho = 0.859, P < 0.0001 rho = 0.718, P < 0.0001- rho = 0.799, P < 0.0001N.S. N.S.N.S. N.S.N.S. N.S.rho = 0.732, P < 0.0001 rho = 0.824, P < 0.0001rho = 0.808, P < 0.0001 rho = 0.835, P < 0.0001rho = 0.836, P < 0.0001 rho = 0.720, P < 0.0001- rho = 0.900, P < 0.0001fluid), MMSE (mini-mental state examination), T-tau (total tau), P-tau181nificant (N.S.) (P-value > 0.05) correlations were not reported.might be a useful as a surrogate biomarker in future cli-nical treatment studies with tau modifying drugs [32].ConclusionsIn summary, we have developed an assay allowing repro-ducible measurement of the level of the presynaptic pro-tein SNAP-25 in CSF samples from individual patients.We demonstrate significantly higher levels of SNAP-25 inCSF samples from patients with prodromal Alzheimer’sdisease and Alzheimer’s disease compared with controls.Our results show that SNAP-25 is a promising novel CSFbiomarker for synapse degeneration in Alzheimer’s dis-ease. This finding could be important for earlier diagnosis,assessment of progression of disease and to monitor drugeffects in treatment trials in neurodegenerative diseases.We also report the identification of previously unknown,truncated soluble forms of SNAP-25 that could beemployed to study the dynamics of SNARE protein pro-cessing and recycling.MethodsHuman brain tissue samplesThe study included autopsy-confirmed patients withAlzheimer’s disease (N = 15) and age-matched controls(N = 15). Brain tissues from the region superior parietalgyrus were analyzed. All brain tissues were obtainedBrinkmalm et al. Molecular Neurodegeneration 2014, 9:53 Page 8 of 13http://www.molecularneurodegeneration.com/content/9/1/53from the Netherlands Brain Bank. Braak and Braakcriteria, which are based on the distribution of neuro-fibrillary tangles, were used to categorize the stage ofAlzheimer’s disease [33]. All Alzheimer’s disease patientsfulfilled Braak stages 5 or 6, while the controls fulfilledBraak stages 0 or 1. Additional file 1: Table S1 shows theclinical and demographic characteristics of the groups.CSF samplesThe exploratory phase of the investigation was performedon pooled decoded CSF samples supplied by the ClinicalNeurochemistry Laboratory, Sahlgrenska University Hos-pital Sweden, from patients who underwent lumbar punc-ture to exclude infectious disorders of the central nervoussystem.The German cohortCSF samples were obtained at the Interdisciplinary Me-mory Clinic of the Department of Geriatric Psychiatry ofthe Clinic of Psychiatry at the Central Institute of MentalHealth, Mannheim from subjects with Alzheimer’s disease(N = 9), prodromal Alzheimer’s disease (N = 7) and non-demented controls (N = 9) (Table 1). Alzheimer’s diseasewas diagnosed according to the NINCDS-ADRDA cri-teria, with all Alzheimer’s disease patients fulfilling thecriteria for probable Alzheimer’s disease [34]. Mild cogni-tive impairment due to Alzheimer’s disease was diagnosedaccording the new research criteria of Albert et al in 2011[35]. Mild cognitive impairment was considered due toprodromal Alzheimer’s disease if additionally, biomarkersof molecular neuropathology of Alzheimer’s disease inCSF were measured positively for Alzheimer’s disease(CSF biomarkers Aβ1-42 ≤450 ng/L; T-tau ≥450 ng/L;P-tau181 ≥61 ng/L) or if there was hippocampal volumereduction or medial temporal atrophy assessed by visualrating (Schelten’s scale >2) measured by an experiencedneuroradiologist. Non-demented controls had variouspsychiatric diagnoses, (including geriatric depression, andschizophrenia), Lumbar puncture in these patients wascarried out for clinical indications, such as excluding or-ganic brain disorder. All were found normal on cognitivescreening tests, all routine CSF analyses were within nor-mal limits and none of the CSF biomarkers were positivefor Alzheimer’s disease.The Swedish cohort ICSF samples were obtained at the Memory Clinic atSkåne University Hospital in Malmö from subjects withAlzheimer’s disease (N = 10) and non-demented controls(N = 6) (Table 1). Subjects diagnosed with Alzheimer’s dis-ease met the DSM-III-R criteria for dementia [36] and thecriteria for probable Alzheimer’s disease, as defined byNINCDS-ADRDA [34]. The non-demented cases exhi-bited cognitive complaints, but did not fulfil the criteriafor dementia. To rule out preclinical Alzheimer’s diseasein the latter group we only included cases with normalCSF Aβ1-42 > 550 ng/L and T-tau <400 ng/L levels.The Swedish cohort IICSF samples from subjects with Alzheimer’s disease(N = 17) and healthy controls (N = 17) were obtainedfrom the Gothenburg mild cognitive impairment studyfor which the diagnostic procedure was described in de-tail previously [37] (Table 1). The diagnosis of dementiawas based on the DSM-III-R criteria [36] together withthe criteria of NINCDS-ADRDA [34] and ICD-10 [38]with regard to Alzheimer’s disease. Controls were not in-cluded if they had subjective or objective signs of a cog-nitive disorder.CSF collectionAll CSF samples were obtained by lumbar puncturethough the L3/L4 interspace. The CSF samples werecentrifuged at 2,000 g for 10 min at room tempera-ture to remove cells and debris, and stored in aliquotsat –80°C pending biochemical analysis.Homogenization of brain tissueThe brain extraction procedure was performed as de-scribed by Öhrfelt et al. with minor modifications [39].Briefly, 100 ± 10 mg of brain tissue was homogenized onice in 1 mL Tris- hydrochloride buffer (10 mM Tris-HCl,pH 6.8) containing complete protease inhibitor (RocheDiagnostics GmBH). Centrifugation of the homogenatewas performed at 31,000 g for 1 h at +4°C and the super-natant was collected (Tris fraction, i.e., soluble fraction).One milliliter of Tris-buffer containing 0.5% Triton X-100(Union Carbide Corporation) with complete protease in-hibitor was added, and the pellet was homogenized on iceand sonicated using a micro-probe sonicator. The cen-trifugation step was repeated and the supernatant wascollected (0.5% Triton fraction, i.e., membrane-boundfraction). The same procedure was repeated by addition ofTris-buffer containing 2% Triton (2% Triton fraction)and complete protease inhibitor, and again by additionof Tris-buffer containing 0.5% sodium dodecyl sulphateand complete protease inhibitor for a final centrifuga-tion at +12°C (SDS fraction, i.e., membrane-raft asso-ciated fraction). All supernatants were aliquoted andstored at –80°C pending analysis. For protein quantita-tion, Protein DC assay (Bio-Rad Laboratories) reagentwas used. This reagent contains a reducing agent and isdetergent compatible.Analysis of CSF biomarkersThe CSF analyses on Aβ1-42, T-tau and P-tau181 levelswere performed using commercially available assays fromBrinkmalm et al. Molecular Neurodegeneration 2014, 9:53 Page 9 of 13http://www.molecularneurodegeneration.com/content/9/1/53Fujirebio (INNOTEST® β-AMYLOID(1-42), INNOTEST®hTAU Ag and INNOTEST® PHOSPHO-TAU(181P).Antibodies and recombinant protein of SNAP-25The following antibodies were used: mouse monoclonalantibody SP12 recognizing SNAP-25 [40,41] and mousemonoclonal antibody SMI81 (Covance) against SNAP-25[25]. The SMI81 antibody recognizes an epitope contai-ning the N-terminally acetylated first 11 amino acids ofbrain SNAP-25 [25]. Recombinant standard protein ofSNAP-25 was purchased from Origene.ImmunoprecipitationThe immunoprecipitation method for brain tissue extractsand CSF samples was performed according to Öhrfeltet al. with minor modifications [39]. Briefly, an aliquot(1 μg) of the mouse monoclonal antibody SP12 (1 g/L) orthe mouse monoclonal antibody SMI81 (1 g/L) or IgG frommurine serum (1 g/L, Sigma-Aldrich) (a negative control),was separately added to 100 μL magnetic DynabeadsM-280 Sheep anti-mouse IgG (Invitrogen Corporation) andincubated 1 h on a rocking platform at room temperature.The beads were washed three times with 1 mL of PBS(10 mM Na-phosphate, 0.15 M NaCl, pH 7.4). Theantibodies were cross-linked using 20 mM dimethyl pimeli-midate dihydrochloride (Sigma-Aldrich) and 0.2 M trie-thanolamine (pH 8.2 Sigma-Aldrich) according to themanufacturer’s product description. The cross-linked beadswere washed two times in PBS and were blocked with Roti-Block (Carl Roth) for 1 h on a rocking platform at roomtemperature. Each brain tissue extract (soluble, membrane-bound, and membrane-raft associated proteins) (26 μg oftotal protein) and CSF samples (German cohort, 890 μL;Swedish cohort I, 700 μL; Swedish cohort II, 600 μL) wereadjusted with 20% Triton and PBS to a final concentrationof 0.2% Triton and a final volume of 1 mL). Samples andmagnetic beads were incubated overnight on a rocking plat-form at +4°C. The magnetic beads/sample solution wastransferred to the KingFisher magnetic particle processor(Thermo Fisher Scientific), tube 1. The following threewash steps (tubes 2-4) were conducted for 10 s in 1 mL ofeach washing buffer: (tube 2) 0.025% Tween 20 in PBS,(tube 3) PBS and (tube 4) 50 mM ammonium hydrogencarbonate (NH4HCO3, pH 8.0). SNAP-25 was then elutedfrom the beads by adding 100 μL 0.5% formic acid (FA)(tube 5) for 4 min. The eluted fractions were transferred to0.5 mL Protein LoBind Tube (Eppendorf AG) and dried ina vacuum centrifuge.Protein digestion and addition of heavy-isotope labeledpeptide standardsBrain tissueThe dried immunoprecipitated brain tissue samples weredissolved in 10 μL 0.1% RapiGest SF Surfactant (Waters)in 50 mM NH4HCO3 1 h in room temperature. Disulfidebonds were reduced by addition of 10 μL 10 mM dithio-threitol (Sigma-Aldrich) in 50 mM NH4HCO3 and incu-bation for 3 min at +90°C. After cooling to roomtemperature, 5 μL 10 mM iodoacetamide (Sigma-Aldrich)in 50 mM NH4HCO3 was added, and the samples wereincubated in the dark at room temperature for 30 min.Digestion was carried out by adding 5 μL trypsin solution(1 μg Sequencing Grade Modified Trypsin [Promega] dis-solved in 0.01% aqueous HCl [0.1 g/L] and diluted to5 mg/L in 50 mM NH4HCO3) and incubating overnightat +37°C. To reduce the amount of RapiGest SF Surfactantin the samples 2 μL 10% aqueous trifluoroacetic acid wasadded (resulting in pH <2). The samples were incubated45 min at +37°C and then centrifuged (16,900 g, 10 min,+4°C). Twenty-five microliters of the resulting supernatantof each sample was carefully transferred to 0.5 mL ProteinLoBind Tubes (Eppendorf AG). A C-terminally isoto-pically labeled peptide, containing U-13C6, U-15 N4-ar-ginine [R] (aa 18-30, ADQLADESLEST[R]) was suppliedby Sigma-Aldrich at over 95% peptide purity as determinedby reversed phase HPLC. The peptide was dissolved anddiluted in 0.1% aqueous FA to a final concentration of5 fmol/μL. A 25 μL aliquot of the reference peptide wasadded to each immunoprecipitated brain homogenatesample after digestion and centrifugation.CSFThe dried immunoprecipitated CSF samples were dis-solved in 5 μL of a mixture of five isotopically labeled pep-tides, containing U-13C6, U-15 N4-arginine [R], U-13C6,U-15 N1-leucine [L] or U-13C6, U-15 N2-lysine [K] andcommon for the N-terminal part of both SNAP-25Aand SNAP-25B. The peptide standard supplied by SigmaAldrich (aa 18-30, see above) was mixed with four Heavy-Peptide FasTrack 1 standards (Thermo Fisher Scientific)(~0.5 mg dissolved in 1 mL MilliQ water) (aa Ac2-16,AEDADMRNE[L]EEMQR; aa 17-31, RADQ[L]ADESLESTRR; aa 18-31 ADQLADESLEST[R][R]; and aa 32-40MLQLVEES[K]) and diluted in 50 mM NH4HCO3to a final concentration of ~100 fmol/μL (aa 18-30)and ~3 ng/μL (FasTrack 1 standards). Reduction of disul-fide bonds (5 μL 10 mM dithiothreitol), alkylation (5 μL10 mM iodoacetamide), and digestion (5 μL 5 mg/Ltrypsin) were performed as described above. To stop theenzymatic activity 4 μL 10% aqueous FA was added.The samples were centrifuged (16,900 g, 10 min, +4°C)and 20 μL of each sample was transferred to LC-vials(SUN-SRi).SRM-MS analysis of immunoprecipitated SNAP-25 frombrain tissueAliquots (25 μL) of the 1:1 mixtures of stable isotope-labeled standard peptide and immunoprecipitated SNAP-25Brinkmalm et al. Molecular Neurodegeneration 2014, 9:53 Page 10 of 13http://www.molecularneurodegeneration.com/content/9/1/53were transferred to LC-vials (SUN-SRi) and analyzed bySRM-MS using an Accela 1250 pump (Thermo FischerScientific) coupled to a triple quadrupole mass spectrom-eter (TSQ Vantage, Thermo Fischer Scientific) with anIonMax source and HESI-II electrospray probe (ThermoFischer Scientific). Mobile phases were 0.1% aqueous FA(v/v) (A) and 0.1% FA in 84% ACN in water (v/v) (B).Samples (20 μL) were loaded directly onto a HypersilGold-C18 column, (length 50 mm, inner diameter2.1 mm, particle size 5 μm [Thermo Fischer Scientific])with 0.1% aqueous FA at 100 μL/min. After 2 min ofloading, the peptides were eluted off the column usingthe following linear gradient steps: 0 min 0%B; 4 min17%B; 12 min 23%B; 15 min 100%B. The global MS pa-rameters were: positive ion mode; spray voltage 3.5 kV;vaporizer temperature +350°C; sheath gas pressure40 psi; auxiliary gas pressure 25 arbitrary units; capillarytemperature +350°C; collision gas pressure 1.9 mTorr.Pinpoint software version 1.3.0 (Thermo Fischer Scientific)was used for method optimization and data processing.LC–MS/MS analysis of immunoprecipitated SNAP-25 frombrain tissueAliquots (25 μL) of the 1:1 mixtures of stable isotopelabeled standard peptide and immunoprecipitatedSNAP-25 protein were transferred to LC-vials (Waters)and analyzed by LC-MS/MS. The LC-MS/MS spectrawere acquired with a electrospray–linear quadrupoleion trap–Fourier transform ion cyclotron resonance(ESI-LQIT–FTICR) mass spectrometer equipped with a7 T magnet (LTQ FT Ultra, Thermo Fischer Scientific)coupled to a multi-dimensional nanoflow chromato-graphy system (Ettan MDLC, GE Healthcare). A Zorbax300 SB-C18 trap column (length 5 mm, inner diameter0.3 mm, particle size 5 μm [Agilent Technologies]) wasused for on-line desalting and a reversed phase Zorbax300 SBC18 column (length 150 mm, inner diameter0.075 mm, particle size 3.5 μm [Agilent Technologies])was used for high-resolution separation. Mobile phaseswere 0.1% aqueous FA (v/v) (A) and 0.1% FA in 84%ACN in water (v/v) (B). The separation was performedat a flow rate of approximately 250 nL/min by applyinga linear gradient of 0-60% B for 50 min. The LTQ FTUltra was set to acquire positive ions and operated inthe data-dependent mode, where a scan cycle consistedof one full scan mass spectrum (m/z 350-1500) acquiredin the FTICR mode (resolution 25,000), followed by tan-dem mass spectrometry (MS/MS) scans acquired inLQIT mode using collision-induced dissociation (CID)and wideband activation. Dynamic exclusion was en-abled with repeat count 2 and exclusion duration 120 s.Isolation width was 3 m/z units, and the normalizedcollision energy to 35. Each scan consisted of threemicroscans.Database search and bioinformatic analysisDatabase searches were submitted to the in-house databaseserver by using Mascot Deamon 2.3.0 (Matrix Science).Database search parameters were; database (UniProtKB_Human 131030), taxonomy (Homo sapiens), enzyme(trypsin), variable modifications (acetyl [N-term], oxida-tion [M], Label:13C(6)15 N(2) [K], and Label:13C(6)15 N(4) [R]), fixed modifications (Carbamidomethyl [C]), massvalues (monoisotopic), peptide mass tolerance (5 ppm),fragment mass tolerance (0.5 Da), and max missed clea-vages (2).Quantification of high mass accuracy precursor ionsPeak detection and integration were performed usingDeCyder 2.0 (GE Healthcare) using processing parame-ters as described in [42]. In-house developed software(Sequence and PeakExtractor) was used for in silico di-gestion and automatic peak mass matching.Top-down-LC-MS/MS analysis of intact SNAP-25fragments from brain tissueSoluble SNAP-25 forms immunoprecipitated with SMI81from three different brain homogenate fractions werepooled in a 0.5 mL Protein LoBind Tube (Eppendorf AG)and dried in a vacuum centrifuge. The sample was dis-solved in 25 μL of 0.1% aqueous FA for 1 h and then cen-trifuged (16,900 g, 10 min, +4°C). The LC-MS/MS spectrawere acquired with the Ettan MDLC/LTQ FT Ultra sys-tem using the same LC settings and columns as for thedigested samples (see above). The LTQ FT Ultra was ope-rated in data-dependent mode with a scan cycle consistingof one full scan mass spectrum acquired in FTICR mode(m/z 500-1,200, resolution 50,000), and one MS/MS scanin FTICR mode (resolution 50,000) using CID. Each scanconsisted of three microscans. Isolation width was 7 m/zunits, and the normalized collision energy 35. To ensureMS/MS acquisition for the peptides of interest, an in-clusion mass list was utilized. Peak picking and chargedeconvolution of acquired spectra were performed usingMascot Distiller (Matrix Science) with processing parame-ters as described in [42]. Database search parameters were;database (UniProtKB_Human 120809), taxonomy (Homosapiens), enzyme (none), variable modifications (oxidation[M]), fixed modifications (acetyl [N-term], carbamido-methyl [C]), mass values (monoisotopic), peptide mass tol-erance (10 ppm), fragment mass tolerance (50 mmu), andinstrument type (FTICR CID Distiller, meaning only singlycharged fragment ions were considered in the databasesearch).HR-SIM-MS analysis of immunoprecipitated SNAP-25from CSFHigh-resolution selected ion monitoring (HR-SIM) ana-lyses were performed on a quadrupole–orbitrap massSNAP-25 peptide Ac-2-16, and the spiked in isotopically labeled peptidesimpairment; MMSE: Mini-mental state examination; ROC: Receiver operatinganalyses; LF, LH, LM, OH, and AW recruited subjects and analyzed clinicaldata and all authors wrote the paper. All authors read and approved theBrinkmalm et al. Molecular Neurodegeneration 2014, 9:53 Page 11 of 13http://www.molecularneurodegeneration.com/content/9/1/53spectrometer Q Exactive (Thermo Fisher Scientific)coupled to an Ultimate 3000 chromatography system(Thermo Fisher Scientific). The samples (15 μL) wereloaded directly onto a Hypersil Gold-C18 column, (seeSRM-MS analysis) with 0.1% aqueous FA at 100 μL/min.Mobile phases were the same as in the SRM-MS analysis.After 2 min of loading, the peptides were eluted off thecolumn using the following linear gradient steps: 0 min0%B; 4 min 13%B; 30 min 17%B; 50 min 26%B; 52 min90%B. The IonMax ion source settings were: spray vol-tage, +4100 V; capillary temperature, +320°C; sheath gaspressure, 25 arbitrary units; auxiliary gas pressure, 10 arbi-trary units; and heater temperature, +300°C. The instru-ment was set to acquire scheduled pairs of SIM scans andsubsequent all ion fragmentation scans in profile modeallowing simultaneous detection of both the SNAP-25peptide and the corresponding isotopically labeled peptidestandard. The settings were common for both scans typesand were as follows: resolution, 70,000; AGC target, 3e6;maximum injection time, 300 ms. Data acquisition andanalysis were performed with Xcalibar software version2.2 SP1.48 (Thermo Fisher Scientific) and Pinpoint 1.3.0.SNAP-25 levels for the different tryptic peptides werecompensated for the different CSF volumes and reportedas the ratio between the peak areas of the endogenouspeptide and the labeled peptide standard multiplied by10,000.Investigation of reproducibilityApproximately 5 pmol each of two isotopically labeled N-terminal SNAP-25 peptides (aa Ac2-16, AEDADM[R]NELEEMQ[R] and aa Ac2-20 AEDADMRNE[L]EEMQRRADQ, FasTrack 1 [Thermo Fisher Scientific]) wereadded to 15 mL CSF. The spiked CSF pool was dividedinto 890 μL aliquots, immunoprecipitated with SMI81and digested as described above. HR-SIM-MS analysis ofeight of the samples was performed on 8 μL injec-tions. The CV of the measured levels was less than 10%(Additional file 1: Table S3).Statistical analysisBecause the distributions of most analytes were notnormal (Shapiro-Wilk test, P <0.05), non-parametric sta-tistics were used for analysis. Data are given as median(inter-quartile range). Differences between more than twogroups were assessed with Kruskal-Wallis test. Statisticallysignificant results (P < 0.05) were followed by Mann-Whitney U-tests to investigate group differences. Sincethere were no significant alterations of the levels of novelSNAP-25 biomarkers, Aβ1-42, and P-tau181 between thedifferent cohorts, it was possible to perform the receiveroperating characteristic (ROC) curve analysis and assesscorrelations in all patients with Alzheimer’s disease andcontrols. ROC curves were performed on each subjectfinal manuscript.AcknowledgementsWe are grateful to Rita Persson for her technical assistance. This work wassupported by grants from the Swedish Brain Power consortium, SwedishAlzheimer Foundation, Swedish Research Council, ALF, the Knut and AliceWallenberg Foundation, Demensfonden, Eivind och Elsa K:son Sylvansstiftelse, the Wolfson Foundation, Märtha och Gustaf Ågrens stiftelse, Gunoch Bertil Stohnes stiftelse, Stiftelsen Gamla Tjänarinnor, Magn. Bergvallscharacteristic; SRM-MS: Selected reaction monitoring–mass spectrometry;SNARE: Soluble N-ethylmaleimide sensitive fusion attachment proteinreceptor; SNAP-25: Synaptosomal-associated protein 25; P-tau181: Tauphosphorylated at threonine 181; T-tau: Total tau.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsAB and AÖ designed and performed the research; AB, GB, WGH, HZ, KB, andAÖ analyzed the data and interpreted results; AÖ performed statisticalAc-2-16[R] and Ac-2-16[L]. Figure S1. Heat map for the relative signalintensities of individual tryptic peptides from SNAP-25B. Figure S2.Tandem mass spectra of three different soluble N-terminal fragments ofSNAP-25 acquired in the FTICR mode.AbbreviationsAβ1-42: Amyloidβ1-42; CSF: Cerebrospinal fluid; CID: Collision-induceddissociation; FA: Formic acid; FTICR: Fourier transform ion cyclotronresonance; HR-SIM-MS: High resolution–selected ion monitoring–massspectrometry; LQIT: Linear quadrupole ion trap; LC-MS/MS: Liquidchromatography–tandem mass spectrometry; MCI: Mild cognitivegroup on the tryptic peptides of SNAP-25 in order to as-sess their diagnostic value. For each tryptic peptide ofSNAP-25 the area under the curve and a 95% confidenceinterval was calculated using GraphPad Prism 5. Thecorrelation coefficients (rho) were calculated using theSpearman two-tailed correlation test. SPSS 20.0 wasemployed for most of the statistical analyses.EthicsThe present study was approved by the Regional EthicsCommittee at the medical faculty Mannheim, Universityof Heidelberg, Germany and Lund University, GothenburgUniversity, Sweden. All patients gave their informed con-sent for research, which was conducted in accordancewith the Helsinki Declaration The ethical principlesabided by Netherlands Brain Bank are found at the web-site: (www.hersenbank.nl).Additional fileAdditional file 1: Table S1. Clinical and demographic characteristics ofthe brain tissue material. Table S2. Soluble SNAP-25 forms identified byLC-MS/MS. Table S3. HR-SIM-MS peak area of the human CSF trypticstiftelse, Svenska Läkaresällskapet, the Canadian Institutes of Health Research(MOP-14037 and CBG-101827), Åhlén-stiftelsen and BMBF BIOMARK-APD(DLR 01ED1203 J).Brinkmalm et al. Molecular Neurodegeneration 2014, 9:53 Page 12 of 13http://www.molecularneurodegeneration.com/content/9/1/53Author details1Institute of Neuroscience and Physiology, Department of Psychiatry andNeurochemistry, Sahlgrenska Academy at the University of Gothenburg,S-431 80 Mölndal, Sweden. 2Department of Psychiatry, University of BritishColumbia, Vancouver, Canada. 3Department of Geriatric Psychiatry, CentralInstitute for Mental Health Mannheim, University of Heidelberg, Mannheim,Germany. 4Clinical Memory Research Unit, Department of Clinical Sciences,Lund University, Lund, Sweden. 5Memory Clinic, Skåne University Hospital,Skåne, Sweden. 6UCL Institute of Neurology, Queen Square, London WC1N3BG, London, UK.Received: 8 July 2014 Accepted: 2 October 2014Published: 23 November 2014References1. 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Brinkmalm G, Portelius E, Ohrfelt A, Mattsson N, Persson R, Gustavsson MK,Vite CH, Gobom J, Mansson J-E, Nilsson J, Halim A, Larson G, Ruetschi U,Zetterberg H, Blennow K, Brinkmalm A: An online nano-LC-ESI-FTICR-MSmethod for comprehensive characterization of endogenous fragmentsfrom amyloid beta and amyloid precursor protein in human and catcerebrospinal fluid. J Mass Spectrom 2012, 47:591–603.doi:10.1186/1750-1326-9-53Cite this article as: Brinkmalm et al.: SNAP-25 is a promising novelcerebrospinal fluid biomarker for synapse degeneration in Alzheimer’sdisease. Molecular Neurodegeneration 2014 9:53.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionBrinkmalm et al. 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