Alpha-, Beta-, and Gamma-synuclein Quantification in Cerebrospinal Fluid by Multiple Reaction Monitoring Reveals Increased Concentrations in Alzheimer′s and Creutzfeldt-Jakob Disease but No Alteration in Synucleinopathies

α-Synuclein (αSyn) is a major constituent of proteinaceous aggregates in neurodegenerative diseases such as Parkinson′s disease (PD) and a potential biomarker candidate for diagnosis and treatment effects. However, studies about αSyn in cerebrospinal fluid (CSF) in diseases are inconsistent and mainly based on immunological assays. Quantitative information about β-synuclein (βSyn) and γ-synuclein (γSyn) in CSF is not available.Here, we present an alternative method for the simultaneous quantification of αSyn, βSyn and γSyn in CSF by multiple reaction monitoring (MRM) with a high sequence coverage (70%) of αSyn to validate previous, ELISA-based results and characterize synucleins in CSF in more detail.The MRM has high sensitivity in the low pg/ml range (3–30pg/ml full-length αSyn) using 200 μl CSF. A high portion of CSF αSyn is present in the N-terminally acetylated form and the concentration of unmodified peptides in the nonamyloid component region is about 40% lower than in the N-terminal region. Synuclein concentrations show a high correlation with each other in CSF (r>0.80) and in contrast to αSyn and γSyn, βSyn is not affected by blood contamination. CSF αSyn, βSyn and γSyn concentrations were increased in Alzheimer′s and Creutzfeldt-Jakob disease but not altered in PD, PD dementia (PDD), Lewy body dementia and atypical parkinsonian syndromes. The ratio βSyn/αSyn was increased in PDD (1.49 ± 0.38, p < 0.05) compared with PD (1.11 ± 0.26) and controls (1.15 ± 0.28). βSyn shows a high correlation with CSF tau concentrations (r = 0.86, p < 0.0001, n = 125).In conclusion, we could not confirm previous observations of reduced αSyn in PD and our results indicate that CSF synuclein concentrations are rather general markers of synaptic degeneration than specific for synucleinopathies. βsyn is an attractive biomarker candidate that might be used as an alternative to or in combination with tau in AD and CJD diagnosis and in combination with αSyn it is a biomarker candidate for PDD. α-Synuclein (αSyn) is a major constituent of proteinaceous aggregates in neurodegenerative diseases such as Parkinson′s disease (PD) and a potential biomarker candidate for diagnosis and treatment effects. However, studies about αSyn in cerebrospinal fluid (CSF) in diseases are inconsistent and mainly based on immunological assays. Quantitative information about β-synuclein (βSyn) and γ-synuclein (γSyn) in CSF is not available. Here, we present an alternative method for the simultaneous quantification of αSyn, βSyn and γSyn in CSF by multiple reaction monitoring (MRM) with a high sequence coverage (70%) of αSyn to validate previous, ELISA-based results and characterize synucleins in CSF in more detail. The MRM has high sensitivity in the low pg/ml range (3–30pg/ml full-length αSyn) using 200 μl CSF. A high portion of CSF αSyn is present in the N-terminally acetylated form and the concentration of unmodified peptides in the nonamyloid component region is about 40% lower than in the N-terminal region. Synuclein concentrations show a high correlation with each other in CSF (r>0.80) and in contrast to αSyn and γSyn, βSyn is not affected by blood contamination. CSF αSyn, βSyn and γSyn concentrations were increased in Alzheimer′s and Creutzfeldt-Jakob disease but not altered in PD, PD dementia (PDD), Lewy body dementia and atypical parkinsonian syndromes. The ratio βSyn/αSyn was increased in PDD (1.49 ± 0.38, p < 0.05) compared with PD (1.11 ± 0.26) and controls (1.15 ± 0.28). βSyn shows a high correlation with CSF tau concentrations (r = 0.86, p < 0.0001, n = 125). In conclusion, we could not confirm previous observations of reduced αSyn in PD and our results indicate that CSF synuclein concentrations are rather general markers of synaptic degeneration than specific for synucleinopathies. βsyn is an attractive biomarker candidate that might be used as an alternative to or in combination with tau in AD and CJD diagnosis and in combination with αSyn it is a biomarker candidate for PDD. α-Synuclein (αSyn) 1The abbreviations used are: αSynα-synucleinAcN-terminal acetylationaCSFartificial CSFADAlzheimer′s diseaseAlbserum albuminα1Chα1-antichymotrypsinα1Trα1-antitrypsinα2HSα2-HS-glycoproteinα2Mα2-macroglobulinβSynβ-synucleinCBScorticobasal syndromeCEcollision energyChPchoroid plexusCJDCreutzfeldt-Jakob diseaseCPceruloplasminCSFcerebrospinal fluidCURcurtain gasISinternal standardγSynγ-synucleinHSAhuman serum albuminHbhemoglobinHbbhemoglobin beta subunitHpxhemopexinIgimmunoglobulinLBDLewy body dementiaLLOQlower limit of quantificationLODlimit of detectionMeOHmethanolMRMmultiple reaction monitoringNACnon-Abeta componentNINCDS-ADRDANational Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders AssociationO-GlcNAcO-linked β-N-acetylglucosaminePDParkinon′s diseasePDDPD dementiaPSAQprotein standard absolute quantificationPSPprogressive supranuclear palsyPTMpost-translational modificationQC samplequality control sampleTEABtriethylammonium bicarbonate. is a small (14 kDa) presynaptic protein and a key player in the pathogenesis of several neurodegenerative diseases such as Parkinson′s disease (PD), PD dementia (PDD), and Lewy body dementia (LBD). None of these diseases is curable to date and diagnosis is based on clinical symptoms (1.McCann H. Stevens C.H. Cartwright H. Halliday G.M. α-Synucleinopathy phenotypes.Parkinsonism Relat. Disord. 2014; 20: S62-S70Abstract Full Text PDF PubMed Scopus (226) Google Scholar). Aggregated αSyn is the main constituent of Lewy bodies which are histopathological hallmarks in the brain of these synucleinopathies. Oligomerization and aggregation of αSyn is neurotoxic and thought to be a causative factor in the neurodegenerative process. Many post-translational modifications (PTMs) have been described for αSyn (e.g. phosphorylation, oxidation) (2.Beyer K. Ariza A. α-Synuclein posttranslational modification and alternative splicing as a trigger for neurodegeneration.Mol. Neurobiol. 2013; 47: 509-524Crossref PubMed Scopus (95) Google Scholar) and there is evidence that PTMs might influence its aggregation and toxic potential (in addition to other mechanisms such as αSyn mutations or metal ion binding) (3.Lashuel H.A. Overk C.R. Oueslati A. Masliah E. The many faces of α-synuclein: from structure and toxicity to therapeutic target.Nat. Rev. Neurosci. 2013; 14: 38-48Crossref PubMed Scopus (1024) Google Scholar). Additionally, shorter forms of αSyn with unknown function can be generated by alternative splicing of the αSyn gene (2.Beyer K. Ariza A. α-Synuclein posttranslational modification and alternative splicing as a trigger for neurodegeneration.Mol. Neurobiol. 2013; 47: 509-524Crossref PubMed Scopus (95) Google Scholar). Although αSyn is a cytoplasmic protein it is also present in the cerebrospinal fluid (CSF) (4.El-Agnaf O.M.A. Salem S.A. Paleologou K.E. Cooper L.J. Fullwood N.J. Gibson M.J. Curran M.D. Court J.A. Mann D.M.A. Ikeda S. Cookson M.R. Hardy J. Allsop D. Alpha-synuclein implicated in Parkinson's disease is present in extracellular biological fluids, including human plasma.FASEB J. 2003; 17: 1945-1947Crossref PubMed Scopus (469) Google Scholar). Because of its importance in the pathogenesis of synucleinopathies, αSyn determination in CSF is a promising biomarker candidate for clinical diagnosis and for the development of αSyn modulating drugs. α-synuclein N-terminal acetylation artificial CSF Alzheimer′s disease serum albumin α1-antichymotrypsin α1-antitrypsin α2-HS-glycoprotein α2-macroglobulin β-synuclein corticobasal syndrome collision energy choroid plexus Creutzfeldt-Jakob disease ceruloplasmin cerebrospinal fluid curtain gas internal standard γ-synuclein human serum albumin hemoglobin hemoglobin beta subunit hemopexin immunoglobulin Lewy body dementia lower limit of quantification limit of detection methanol multiple reaction monitoring non-Abeta component National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association O-linked β-N-acetylglucosamine Parkinon′s disease PD dementia protein standard absolute quantification progressive supranuclear palsy post-translational modification quality control sample triethylammonium bicarbonate. Many studies investigated CSF αSyn concentrations in neurodegenerative diseases, especially PD as the most common synucleinopathy (for a review, see ref (5.Parnetti L. Cicognola C. Eusebi P. Chiasserini D. Value of cerebrospinal fluid α-synuclein species as biomarker in Parkinson's diagnosis and prognosis.Biomark. Med. 2016; 10: 35-49Crossref PubMed Scopus (44) Google Scholar)). Most studies observed slightly reduced αSyn concentrations in PD, although results are inconsistent. Additionally, the reported small alterations seem not to be of diagnostic relevance (5.Parnetti L. Cicognola C. Eusebi P. Chiasserini D. Value of cerebrospinal fluid α-synuclein species as biomarker in Parkinson's diagnosis and prognosis.Biomark. Med. 2016; 10: 35-49Crossref PubMed Scopus (44) Google Scholar). To date, the method of choice for αSyn determination are immunoassays but antibodies and platforms vary considerably. This led to large concentration differences between studies and hampers the interpretation of inconsistent results. These differences also raise concern about the selectivity of the assays and, thus, an alternative method is needed to confirm previous observation about CSF αSyn concentrations. The other members of the synuclein protein family, β-synuclein (βSyn) and γ-synuclein (γSyn), are less well studied although they are present in proteinaceous aggregates in some neurodegenerative diseases (6.Galvin J.E. Uryu K. Lee V.M. Trojanowski J.Q. Axon pathology in Parkinson's disease and Lewy body dementia hippocampus contains alpha-, beta-, and gamma-synuclein.Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 13450-13455Crossref PubMed Scopus (368) Google Scholar) and there is evidence for a strong interaction of αSyn and βSyn (7.Hashimoto M. Rockenstein E. Mante M. Mallory M. Masliah E. beta-Synuclein inhibits alpha-synuclein aggregation: a possible role as an anti-parkinsonian factor.Neuron. 2001; 32: 213-223Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). βSyn has been shown to be neuroprotective and inhibits αSyn aggregation (7.Hashimoto M. Rockenstein E. Mante M. Mallory M. Masliah E. beta-Synuclein inhibits alpha-synuclein aggregation: a possible role as an anti-parkinsonian factor.Neuron. 2001; 32: 213-223Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). γSyn aggregation is also associated with widespread neurodegeneration (8.Ninkina N. Peters O. Millership S. Salem H. van der Putten H. Buchman V.L. Gamma-synucleinopathy: neurodegeneration associated with overexpression of the mouse protein.Hum. Mol. Genet. 2009; 18: 1779-1794Crossref PubMed Scopus (88) Google Scholar). Both proteins are present in CSF (9.Guldbrandsen A. Vethe H. Farag Y. Oveland E. Garberg H. Berle M. Myhr K-M Opsahl J.A. Barsnes H. Berven F.S. In-depth characterization of the cerebrospinal fluid (CSF) proteome displayed through the CSF proteome resource (CSF-PR).Mol. Cell. Proteomics. 2014; 13: 3152-3163Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Only a single study investigated γSyn concentrations in CSF of dementia patients by a qualitative Western blot and observed an increase in Alzheimer′s disease (AD) and LBD (10.Mukaetova-Ladinska E.B. Milne J. Andras A. Abdel-All Z. Cerejeira J. Greally E. Robson J. Jaros E. Perry R. McKeith I.G. Brayne C. Xuereb J. Cleghorn A. Doherty J. McIntosh G. Milton I. Alpha- and gamma-synuclein proteins are present in cerebrospinal fluid and are increased in aged subjects with neurodegenerative and vascular changes.Dement. Geriatr. Cogn. Disord. 2008; 26: 32-42Crossref PubMed Scopus (33) Google Scholar) but this was not validated with further studies and information about other neurodegenerative diseases is missing. CSF βSyn has not been investigated in neurological disorders so far. The determination of βSyn and γSyn in CSF and their relation to αSyn would help to clarify their role in neurodegenerative diseases and the ratio of synuclein protein concentrations in CSF might be more meaningful biomarker candidates than each of the proteins alone. Multiple reaction monitoring mass spectrometry (MRM) can be used for accurate, absolute quantification of proteins using stable-labeled protein standards (protein standard absolute quantification, PSAQ) (11.Brun V. Dupuis A. Adrait A. Marcellin M. Thomas D. Court M. Vandenesch F. Garin J. Isotope-labeled protein standards: toward absolute quantitative proteomics.Mol. Cell. Proteomics. 2007; 6: 2139-2149Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar) and is an excellent alternative to immunoassays for quantification of αSyn with a high selectivity and the ability for multiplexing, i.e. simultaneous quantification of βSyn and γSyn. MRM has already successfully been applied for the determination of biomarker candidates in CSF (12.Oeckl P. Steinacker P. von Arnim C.A.F. Straub S. Nagl M. Feneberg E. Weishaupt J.H. Ludolph A.C. Otto M. Intact protein analysis of ubiquitin in cerebrospinal fluid by multiple reaction monitoring reveals differences in Alzheimer′s disease and frontotemporal lobar degeneration.J. Proteome Res. 2014; 13: 4518-4525Crossref PubMed Scopus (28) Google Scholar). In addition, it allows a more detailed characterization of the whole protein regarding truncations or PTMs by analyzing several peptides across the protein sequence after proteolytic digestion and αSyn PTMs are in discussion as promising biomarker candidates in synucleinopathies (13.Schmid A.W. Fauvet B. Moniatte M. Lashuel H.A. Alpha-synuclein post-translational modifications as potential biomarkers for Parkinson disease and other synucleinopathies.Mol. Cell. Proteomics. 2013; 12: 3543-3558Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Detailed protein characterization by MRM has recently successfully been shown for the tau protein in CSF, a biomarker used in the diagnostics of AD, which seems to be predominantly N- and C-terminally truncated in CSF (14.Barthélemy N.R. Fenaille F. Hirtz C. Sergeant N. Schraen-Maschke S. Vialaret J. Buée L. Gabelle A. Junot C. Lehmann S. Becher F. Tau Protein Quantification in Human Cerebrospinal Fluid by Targeted Mass Spectrometry at High Sequence Coverage Provides Insights into Its Primary Structure Heterogeneity.J. Proteome Res. 2016; 15: 667-676Crossref PubMed Scopus (72) Google Scholar). However, it was not possible so far to quantitatively measure αSyn in CSF by MRM in a useful sample volume because of the low concentration in the pg/ml range. We present here an MRM method for the simultaneous and absolute quantification of unmodified αSyn, βSyn, γSyn and hemoglobin in the low pg/ml range in 200 μl CSF using a stable-labeled protein standard (αSyn) and stable-labeled peptides (βSyn, γSyn, PTMs) as internal standards. Seven of eight possible, unmodified tryptic peptides of αSyn are included covering 70% of the αSyn sequence for a more detailed characterization of αSyn. In addition, several PTMs are included for αSyn (Ser87P, Ser87O-GlcNAc, Thr54P, Thr54O-GlcNAc, N-terminal acetylation) as well as two proteotypic peptides for the αSyn splice variants αSyn126 and αSyn112 (Fig. 1). We used the method to characterize synucleins in CSF of patients without neurodegenerative diseases and compared the results with ELISA data. We then measured synucleins in a panel of neurodegenerative diseases including PD, PDD, LBD, progressive supranuclear palsy (PSP), corticobasal syndrome (CBS), AD and Creutzfeldt-Jakob disease (CJD) to validate previous results of CSF αSyn with immunoassays and to gain new information about βSyn, γSyn and the αSyn peptide pattern in CSF. Recombinant full-length αSyn (purity >95%) was purchased from AJ Roboscreen GmbH (Leipzig, Germany) and the exact protein concentration was determined by amino acid analysis (Alphalyse A/S, Odense, Denmark). Full-length βSyn, γSyn and 15N-labeled αSyn (all with purity >95%) were from rPeptide (Bogart, GA) and exact βSyn concentration was determined using αSyn MRM of common peptides. Synthetic peptides (see supplemental Table S1) were purchased from Thermo Fisher Scientific. Trypsin/LysC Mix was from Promega GmbH, triethylammonium bicarbonate (TEAB), ammonium hydroxide solution (LC-MS grade) and human serum albumin (HSA, #A3782) from Sigma, solid phase cation extraction disks from 3M (#2251, St. Paul, MN) and artificial CSF (aCSF) from EcoCyte Bioscience (Austin, TX). All LC solvents were of LC-MS grade and purchased from Thermo Fisher Scientific (DMSO, formic acid, TFA) or Carl Roth GmbH, Karlsruhe, Germany (ACN, methanol (MeOH), water). Proteins and peptides were dissolved in LC-MS water at concentrations of 100–500 μg/ml, aliquoted in protein low binding tubes and stored at −80 °C. Calibration standards and QC samples were prepared freshly in aCSF containing 200 μg/ml HSA for each analytical sequence using recombinant αSyn, βSyn, γSyn, and synthetic peptides for sequences with PTMs and splice variants (see supplemental Table S1). Concentrations of the calibration standards and low, medium and high QC samples covered a range of 1.5–1310 pm (αSyn, βSyn) and 40–2000 pm (γSyn) and are listed in detail in supplemental Table S1. In addition, an unspiked CSF QC sample was included in each sequence. CSF samples were thawed on ice and 200 μl of CSF, calibration standard or QC sample were mixed with 40 μl of internal standard (IS) solution (containing labeled peptides and 15N-αSyn in 0.5 m TEAB, see supplemental Table S1) and 12 μl of Trypsin/LysC solution (0.1 μg/μl in 100 mm TEAB) in protein low binding tubes (Sarstedt, Nümbrecht, Germany). Samples were digested for 16h at 27 °C. After addition of 700 μl water and 100 μl 10% TFA, tryptic peptides were captured with STAGE-tips (15.Rappsilber J. Mann M. Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.Nat. Protoc. 2007; 2: 1896-1906Crossref PubMed Scopus (2570) Google Scholar) containing solid phase cation extraction disks (activated with ACN), washed with 0.2% TFA and eluted into 24-Well PCR plates with increasing concentrations of ammonium acetate in 20% ACN/0.5% formic acid (75 mm →fraction 1, 125 mm →fraction 2, 200 mm →fraction 3) and finally with 5% ammonium hydroxide/80% ACN (fraction 4). Fractions were vacuum dried and redissolved in 25 μl of 0.1% TFA/6% ACN (fraction 1 and 3), 0.5% TFA/6% ACN (fraction 2) and 0.1% TFA/4% ACN (fraction 4) by thorough mixing and sonication, centrifuged and stored in the autosampler at 4 °C. Serum (20 μl) was diluted with 180 μl aCSF and prepared as described for CSF. Samples were analyzed using an Agilent 1260 HPLC pump (Santa Clara, CA), Eksigent microLC200, and AB Sciex QTRAP6500 mass spectrometer (both AB Sciex, Framingham, MA) in positive ionization mode. Twenty microliters of sample were loaded on a C18 PepMap100, 5 μm, 0.3 × 5 mm trap column (Thermo Fisher Scientific) with mobile phase A: 0.05% TFA, and mobile phase B: 0.05% TFA in MeOH. Afterward, peptides were separated on an Eksigent HALO Fused-core C18, 2.7 μm, 0.5 × 100 mm column at 40 °C with mobile phase A: 4% DMSO/0.1% formic acid, and mobile phase B: 4% DMSO/96% ACN/0.1% formic acid (see supplemental Table S2 for gradient settings). The analytical column was connected to the QTRAP6500 with a 25 μm electrode and data were acquired in scheduled MRM mode (retention time window 40–120s, scan time 0.2–0.4s, dwell weight: 0.2 for labeled peptides, 1.0 for others). The ion source settings were as follows: 4900–5500 V, 175 °C, curtain gas (CUR) 30psi, nebulizer gas (GS1) 20–40psi, GS2 30psi and CAD gas high. Transitions used and individual MS settings are described in Table I and supplemental Table S3. Two or three transitions per peptide were acquired and the correct transition pattern of each peptide was verified in all samples using Skyline software 3.1 (16.MacLean B. Tomazela D.M. Shulman N. Chambers M. Finney G.L. Frewen B. Kern R. Tabb D.L. Liebler D.C. MacCoss M.J. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments.Bioinformatics. 2010; 26: 966-968Crossref PubMed Scopus (2964) Google Scholar).Table IMS parameters and chromatographic characteristics. CE: collision energy, RT: retention time, z: charge statePeptide sequence (position)ProteinPrecursor mass (labeled peptide)zProduct ionCE (V)FractionRT (min)MDVFMK (1–6)αSyn385.7 (389.2)2+y5, y319, 2345.1(1–6)βSynAc-MDVFMK (1–6)αSyn812.4 (820.4)1+b3, y349, 4729.2(1–6)βSynEGVVAAAEK (13–21)αSyn437.2 (442.2)2+y5, y6, y318, 20, 1823.5(13–21)βSynQGVAEAAGK (24–32)αSyn415.7 (421.2)2+y6, y421, 2122.1EGVLYVGSK (35–43)αSyn476.3 (481.3)2+y5, y320, 2134.4(35–43)βSynEGVVHGVATVAEK (46–58)αSyn648.4 (656.3)2+y8, y935, 3443.4EGVVHGVAT(Phospho)VAEK (46–58)αSyn688.3 (692.3)2+b5, y8, y437, 35, 3936.1EGVVHGVAT(O-GlcNAc)VAEK (46–58)αSyn749.9 (753.9)2+b5, b7, y8, 204.1 (oxonium ion)38, 46, 35, 3833.1EQVTNVGGAVVTGVTAVAQK (61–80)αSyn964.5 (976.5)2+y10, y9, y1144, 44, 4427.9TVEGAGSIAAATGFVK (81–96)αSyn739.9 (748.4)2+y8, y11, y734, 34, 3436.7TVEGAGS(Phospho)IAAATGFVK (81–96)αSyn779.9 (785.9)2+Y8, y7, y142+34, 34, 2917.8TVEGAGS(O-GlcNAc)IAAATGFVK (81–96)αSyn841.4 (847.4)2+y8, y7, y6, 204.1 (oxonium ion)43, 46, 49, 2736.3EGYQDYEPEA (103–112)αSyn1121200.5 (1206.5)1+b7, b6, y752, 52, 5915.3EGVLYVVAEK (35–44)αSyn126553.8 (557.8)2+y6, y7, y423, 25, 2836.0EGVVQGVASVAEK (46–58)βSyn636.8 (640.9)2+y8, y9, y1030, 28, 2827.0EQASHLGGAVFSGAGNIAAATGLVK (61–85)βSyn776.1 (778.8)3+Y8, b172+31, 2748.1ENVVQSVTSVAEK (46–58)γSyn695.4 (699.4)2+y8, y1034, 3427.2TVEEAENIAVTSGVVR (81–96)γSyn837.4 (842.4)2+y8, y746, 3736.0VNVDEVGGEALGR (19–31)Hbb657.8 (662.8)2+y7, y830, 3126.9 Open table in a new tab In each analytical sequence, calibration standards, QC samples and a blank sample (aCSF+HSA) with and without IS were analyzed in duplicate (one at the beginning and one at the end of the sequence). The order of CSF samples (single measurement) was defined by systematic randomization. Intra-assay precision (%CV) was determined by analysis of four CSF-QC samples in a single run and inter-assay precision by analysis of duplicate CSF-QC samples in four independent runs. The lower limit of quantification (LLOQ) was defined as the lowest concentration with a CV and deviation of ≤20% and the limit of detection (LOD) with a signal-to-noise ratio of 3. Stability of synucleins in CSF was tested by incubation of CSF at bench-top conditions (RT), on ice and with different freeze-thaw cycles (thawing for at least 2 h, freezing for at least 12 h). Dilution stability was determined by dilution of CSF with aCSF up to fourfold. The IS-normalized peak area was used for quantification (synucleins) and all transitions from a single peptide were summed up. Peptide concentrations were calculated based on the calibration curve (weighting 1/x2) using Analyst software 1.6.2 (AB Sciex). Total αSyn and γSyn concentration was calculated as the mean concentration of all proteotypic peptides (see Fig. 1). Concentration of the Hbb (hemoglobin beta subunit) peptide was determined using the IS peak area for one-point calibration and total hemoglobin (Hb) concentration was calculated assuming a 1:1 ratio of the Hb alpha and beta subunit. The Hbb IS was added to samples in a final concentration of 200 ng/ml to increase accuracy at this cut-off. CSF samples with a hemoglobin concentration >200 ng/ml were ruled out for αSyn and γSyn analysis as recommended (17.Hong Z. Shi M. Chung K.A. Quinn J.F. Peskind E.R. Galasko D. Jankovic J. Zabetian C.P. Leverenz J.B. Baird G. Montine T.J. Hancock A.M. Hwang H. Pan C. Bradner J. Kang U.J. Jensen P.H. Zhang J. DJ-1 and alpha-synuclein in human cerebrospinal fluid as biomarkers of Parkinson's disease.Brain. 2010; 133: 713-726Crossref PubMed Scopus (503) Google Scholar). CSF and serum samples for αSyn ELISA (Covance #SIG-38974) measurements were diluted 1:20 and 1:200, respectively, and tau concentration in CSF was determined with a commercial ELISA (Fujirebio). All measurements were performed according to the manufacturer's instructions. Patients were enrolled at the Ulm University Hospital, Department of Neurology. Characteristics of patients are depicted in supplemental Table S4. Control patients had no neurodegenerative disease and CSF was collected to rule out acute or chronic inflammation of the brain. PD patients were diagnosed according to accepted criteria (18.Hughes A.J. Daniel S.E. Kilford L. Lees A.J. Accuracy of clinical diagnosis of idiopathic Parkinson's disease: a clinico-pathological study of 100 cases.J. Neurol. Neurosurg. Psychiatry. 1992; 55: 181-184Crossref PubMed Scopus (8449) Google Scholar), PDD and LBD according to (19.Emre M. Aarsland D. Brown R. Burn D.J. Duyckaerts C. Mizuno Y. Broe G.A. Cummings J. Dickson D.W. Gauthier S. Goldman J. Goetz C. Korczyn A. Lees A. Levy R. Litvan I. McKeith I. Olanow W. Poewe W. Quinn N. Sampaio C. Tolosa E. Dubois B. Clinical diagnostic criteria for dementia associated with Parkinson's disease.Mov. Disord. 2007; 22 (quiz 1837): 1689-1707Crossref PubMed Scopus (2062) Google Scholar, 20.McKeith I.G. Dickson D.W. Lowe J. Emre M. O'Brien J.T. Feldman H. Cummings J. Duda J.E. Lippa C. Perry E.K. Aarsland D. Arai H. Ballard C.G. Boeve B. Burn D.J. Costa D. Del Ser T. Dubois B. Galasko D. Gauthier S. Goetz C.G. Gomez-Tortosa E. Halliday G. Hansen L.A. Hardy J. Iwatsubo T. Kalaria R.N. Kaufer D. Kenny R.A. Korczyn A. Kosaka K. Lee V.M.Y. Lees A. Litvan I. Londos E. Lopez O.L. Minoshima S. Mizuno Y. Molina J.A. Mukaetova-Ladinska E.B. Pasquier F. Perry R.H. Schulz J.B. Trojanowski J.Q. Yamada M. Diagnosis and management of dementia with Lewy bodies: third report of the DLB Consortium.Neurology. 2005; 65: 1863-1872Crossref PubMed Scopus (4132) Google Scholar). Diagnosis of PSP and CBS followed the criteria of (21.Litvan I. Agid Y. Calne D. Campbell G. Dubois B. Duvoisin R.C. Goetz C.G. Golbe L.I. Grafman J. Growdon J.H. Hallett M. Jankovic J. Quinn N.P. Tolosa E. Zee D.S. Clinical research criteria for the diagnosis of progressive supranuclear palsy (Steele-Richardson-Olszewski syndrome): report of the NINDS-SPSP international workshop.Neurology. 1996; 47: 1-9Crossref PubMed Scopus (2099) Google Scholar, 22.Armstrong M.J. Litvan I. Lang A.E. Bak T.H. Bhatia K.P. Borroni B. Boxer A.L. Dickson D.W. Grossman M. Hallett M. Josephs K.A. Kertesz A. Lee S.E. Miller B.L. Reich S.G. Riley D.E. Tolosa E. Tröster A.I. Vidailhet M. Weiner W.J. Criteria for the diagnosis of corticobasal degeneration.Neurology. 2013; 80: 496-503Crossref PubMed Scopus (1000) Google Scholar). AD patients fulfilled the NINCDS-ADRDA (National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association) criteria and CJD was diagnosed according to the WHO consensus criteria (23..World Health Organization (1998) Consensus on criteria for sporadic CJD.Google Scholar). All patients or their relatives gave written informed consent to participate in the study and the collection and analysis of CSF and serum samples was approved by the Ethics Committee of Ulm University. CSF was collected by lumbar puncture at the Ulm University Hospital, Department of Neurology. Samples were centrifuged and stored at −80 °C within 2 h according to local SOPs and standard CSF parameters were determined (24.Jesse S. Brettschneider J. Süssmuth S.D. Landwehrmeyer B.G. von Arnim C.A.F. Ludolph A.C. Tumani H. Otto M. Summary of cerebrospinal fluid routine parameters in neurodegenerative diseases.J. Neurol. 2011; 258: 1034-1041Crossref PubMed Scopus (61) Google Scholar). Statistical analysis was performed using GraphPad Prism 5.0. Disease groups were compared by Kruskal-Wallis test and Dunn′s post hoc test. Correlation analysis was performed using Spearman′s rank correlation coefficient. All theoretical tryptic peptides of αSyn except the C-terminal peptide (see discussion section) were selected for the method to cover the largest part possible (about 70%) of the αSyn sequence. Proteotypic peptides for βSyn and γSyn and the optimal charge state of all peptides were selected based on the observed sensitivity and selectivity. MS parameters were optimized by direct infusion of tryptic peptides and are shown in supplemental Table S3. Fig. 1 gives an overview of selected peptides and their relation to the amino acid sequence and other synuclein variants. To optimize LC conditions we tested different column temperatures, flow rates and compositions of the mobile phases using ACN, MeOH, formic acid, and TFA. The settings were optimized for each sample fraction and are given in supplemental Table S2. The high dynamic range of protein concentrations in CSF significantly hampers the detection of low abundance proteins such as αSyn by LC-MS/MS and we tested several approaches to modify the digestion protocol in favor of synucleins. The synuclein proteins do not contain Cys-residues and a reduction and alkylation step is not necessary. Omitting reduction and alkylation increased sensitivity for αSyn peptides and markedly decreased intensity of selected HSA peptides (10–1000x, label-free estimation). Using a digestion temperature of 27 °C instead of 37 °C and using trypsin/LysC instead of trypsin alone also improved the sensitivity for synucleins. Heating or addition of small amount of ACN (5–10%) to samples before digestion reduced sensitivity for synucleins but increased the intensity of HSA peptides. Transitions of peptides for detection/quantification were selected based on their sensitivity and selectivity examined