Deep Dive on the Proteome of Human Cerebrospinal Fluid: A

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Deep Dive in the Proteome of Human Cerebrospinal Fluid: A Valuable Data Resource for Biomarker Discovery and Missing Protein Identification Charlotte Macron, Lydie Lane, Antonio Núñez Galindo, and Loïc Dayon J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00300 • Publication Date (Web): 20 Aug 2018 Downloaded from http://pubs.acs.org on August 21, 2018

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Journal of Proteome Research

Deep Dive in the Proteome of Human Cerebrospinal Fluid: A Valuable Data Resource for Biomarker Discovery and Missing Protein Identification Charlotte Macron1, Lydie Lane2,3, Antonio Núñez Galindo1 and Loïc Dayon1, *

1

Proteomics, Nestlé Institute of Health Sciences, 1015 Lausanne, Switzerland

2

CALIPHO Group, SIB-Swiss Institute of Bioinformatics, CMU, rue Michel-Servet 1, 1211 Geneva 4,

Switzerland 3

Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, rue

Michel-Servet 1, 1211 Geneva 4, Switzerland *To whom correspondence should be addressed Corresponding Author Dr. Loïc Dayon Nestlé Institute of Health Sciences EPFL Innovation Park, Bâtiment H 1015 Lausanne Switzerland Email: [email protected] Fax: +41 21 632 6499

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Abstract

Cerebrospinal fluid (CSF) is a body fluid of choice for biomarker studies of brain disorders but remains relatively under-studied compared to other biological fluids such as plasma, partly due to the more invasive means of its sample collection. The present study establishes an in-depth CSF proteome through the analysis of a unique CSF sample from a pool of donors. After immuno-affinity depletion, the CSF sample was fractionated using off-gel electrophoresis and analyzed with liquid chromatography tandem mass spectrometry (MS) using the latest generation of hybrid Orbitrap mass spectrometers. The shotgun proteomic analysis allowed the identification of 20689 peptides mapping on 3379 proteins. To the best of our knowledge, the obtained dataset constitutes the largest CSF proteome published so far. Among the CSF proteins identified, 34% correspond to genes whose transcripts are highly expressed in brain according to the Human Protein Atlas. The principal Alzheimer disease biomarkers (e.g., tau protein, amyloid-β, apolipoprotein E and neurogranin) were detected. Importantly, our dataset significantly contributes to the Chromosome-centric Human Proteome Project (C-HPP), and 12 proteins considered as missing are proposed for validation in accordance with the HPP guidelines. Of these 12, 8 proteins are based on 2 to 6 uniquely-mapping peptides from this CSF analysis, and 4 match a new peptide with a “stranded” single peptide in PeptideAtlas from earlier CSF studies. The MS proteomic data are available to the ProteomeXchange Consortium (http://www.proteomexchange.org/)

with

the

dataset

identifier

[email protected], Password: 1iOqr37H).


PXD009646

(Username:

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Keywords: augurin; brain; cerebrospinal fluid; Human Proteome Project; mass spectrometry; proteomics; missing proteins; tandem mass tag

Introduction Biomarker discovery is an active area of research and scientific investigations. The use of biomarkers is for instance crucial for early pathology detection, treatment and monitoring of diseases. Body fluids represent samples of choice in the field since they are generally obtained in a less invasive way than tissue biopsies. For clinical applications, it is indeed key that sample collection is as simple, accessible and reproducible as possible. In that regard, blood remains the reference sample. As shown in Figure 1, the number of scientific publications dealing with the blood plasma proteome has grown exponentially these past 20 years, while the number of publications related to the cerebrospinal fluid (CSF) proteome has remained ten times lower (e.g., 407 scientific articles published in 2017 referred to “blood proteome” while only 40 related to “CSF proteome”).



Figure 1: Evolution of the number of publications for “blood+proteome” and “cerebrospinal fluid/CSF+proteome” over the years. The data were extracted from PubMed on 2018/01/22.

Several factors can explain this lower representation of CSF in proteomic studies. Compared to plasma, CSF sample collection is more invasive as it requires lumbar puncture. One other challenge encountered with CSF is the lower total protein concentration in the fluid compared to plasma and serum (i.e., 0,2% of blood total protein1). CSF also contains a complex mixture of proteins with a high dynamic concentration range, which complicates its analysis1; there are eight orders of magnitude between albumin (i.e., dg/L range), the most abundant CSF protein, and the less abundant proteins (i.e., ng/L range)2 in CSF. Lastly, CSF analysis relevance is mainly limited to brain disorders, damages and diseases. However, CSF constitutes a very precious matrix for the diagnosis of various neurological diseases such as Alzheimer disease (AD)3 and Parkinson disease (PD)4, in particular. This colorless body fluid produced in the choroid plexuses of the ventricles of the brain surrounds the central nervous system (CNS)1. CSF is the only body fluid in direct contact with the brain and has exchanges with blood via the brain-blood barrier. CSF, as a window into the brain, is therefore a useful neurobiological biomarker source for clinical studies. The exploration of the CSF proteome started in the 2000s using two-dimensional (2D) gel electrophoresis and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis and resulted in the identification of 21 proteins5 (Table 1). Two years later, Sickmann et al. repeated the experiment and multiplied by a factor of four the number of identified proteins in CSF 6. Several methods were used to fractionate/decomplexify


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CSF samples as the combination of depletion and capillary electrophoresis7 or the enrichment of glycoproteins8; but the number of proteins identified in CSF remained under 800 for a few years. From 2010, jointly with the development of mass spectrometers with improved speed, resolution and accuracy, increased numbers of proteins have been identified in CSF. A first large dataset of 1212 proteins was obtained by Mouton-Barbosa et al.9 using the combinatorial peptide ligand library technology to reduce the dynamic range of proteins, and MS with a linear trap quadrupole (LTQ)-Orbitrap. The same year, Schutzer et al.10 reported on a method combining immunodepletion and strong cation-exchange (SCX) followed by MS analysis with an LTQOrbitrap; this work resulted in the largest dataset obtained at that time, with 2630 proteins identified in CSF. Guldbrandsen et al.11 then identified 3081 proteins using three different sample preparation methods, and in 2015, Zhang et al.2 further increased the number of identified CSF proteins, using depletion, high-pH reversed-phase (RP) high performance liquid chromatography (HPLC) fractionation, and quadrupole (Q)-TOF analysis. More recently, 3006 proteins were obtained in CSF by analyzing more than one hundred fractions of CSF (from patients with multiple sclerosis and controls) after depletion and glycoprotein enrichment12. Table 1 recapitulates a selection of proteomic studies of CSF samples from “healthy” donors.

Table 1: Proteomic studies of CSF from “healthy” donors since 2000. This table summarizes the sample preparation and analysis method of CSF samples, for a selection of scientific articles published since 2000. In 15 years, the number of identified proteins in CSF has increased from 21 to more than 3000. Paper Sickmann et al. Yuan et al.

13

5

Year

Sample preparation

Instrument

2000

2D-gel electrophoresis

MALDI-TOF

21

2002

Protein precipitation or Bio spin column and 2D-gel electrophoresis

MALDI-TOF

22


Proteins


Sickmann et al.

6

14

Finehout et al.

Maccarrone et al. Xu et al.

15

16

Pan et al.

2002


MALDI-TOF

2004


MALDI-TOF

2004 2006

8

Zougman et al.

17

Mouton-Barbosa et 9 al. 10 Schutzer et al. Guldbrandsen et al.

2

Begcevic et al. 1 2

82

2D-LC ion trap (IT)

1

347

2D-LC IT1 2D-LC LTQ-Fourier transform ion cyclotron resonance (FT-ICR) 2D-LC IT1

466

798 1212 2630

Isolation of N-linked deglycosylated peptides/extraction of glycoproteins

2008

SDS-PAGE

2010

Peptide ligand library and SDS-PAGE

1D-LC LTQ-Orbitrap/LTQFT-ICR 1D-LC LTQ-Orbitrap

2010

Immunodepletion and SCX-RP-HPLC

1D-LC LTQ-Orbitrap

2014

2015 18

Immunodepletion and 2D-gel electrophoresis Sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) and SCX-RP-LC

70

2006

11

Zhang et al.

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2016

SDS-PAGE and mixed mode reversed phase-anion exchange for mapping the global CSF proteome, and hydrazidebased glycopeptide capture for mapping glycopeptides Immunodepletion and high-pH RP-LC SCX-LC

1D-LC LTQ-Orbitrap

1D-LC Q-TOF3 1D-LC- Q-Orbitrap

2

608 216

3081

3256 4

2615

LCQ DECA XP PLUS LTQ-Orbitrap Velos Pro

3

Triple TOF 5600

4

Q Exactive Plus Orbitrap

The progresses accomplished in the past years for comprehensively characterizing the CSF proteome with MS have been considerable. The above-mentioned proteomic studies were achieved from multiple CSF samples prepared with various protocols and analyzed in replicates (Table 1). In the present work, we aimed at deeply studying the CSF proteome from a single sample source using a unique and simple protocol. We analyzed a commercial pool of “normal” CSF samples; the pooled CSF sample was depleted of abundant proteins, labeled with tandem mass tag (TMT) and fractionated in 24 fractions using off-gel electrophoresis (OGE). Each fraction was analyzed independently with RP-LC tandem MS (MS/MS) on the latest generation of hybrid Orbitrap instruments, i.e., the Orbitrap Fusion Lumos Tribrid mass spectrometer. The


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obtained dataset was investigated for the presence of “missing” proteins (MPs), which is one key goal of the Chromosome-Centric Human Proteome Project (C-HPP)19.

Experimental Section Material A commercial pool of “normal” CSF samples (70 mL) was purchased from Analytical Biological Services (Wilmington, DE). The samples constituting this pool were remnant samples collected from individuals for various testing. Each donor gave informed consent. The “normal” CSF used was a pool of CSF samples from donors who were tested for CNS pathologies like meningitis but revealed negative for the investigated indication. The local institutional ethical committee board approved the clinical protocol. Iodoacetamide (IAA), tris (2-carboxyethyl) phosphine hydrochloride (TCEP), triethylammonium hydrogen carbonate buffer (TEAB) (1 M, pH 8.5), sodium dodecyl sulfate (SDS), and β-lactoglobulin (LACB) from bovine milk were purchased from Sigma (St. Louis, MO). Formic acid (FA, 99%) and CH3CN were from BDH (VWR International, Ltd., Poole, UK). Hydroxylamine solution 50 wt% in H2O (99.999%) was acquired from Aldrich (Milwaukee, WI). H2O (18.2 MΩ·cm at 25 °C) was obtained from a Milli-Q apparatus (Millipore, Billerica, MA). Trifluoroacetic acid Uvasol was sourced from Merck Millipore (Billerica, MA). The 6-plex TMTs isobaric label kits were purchased from Thermo Scientific (Rockford, IL). Sequencing-grade modified trypsin/Lys-C was procured from Promega (Madison, WI). For immuno-affinity depletion of 14 highly abundant proteins from human biological fluids, multiple affinity removal system (MARS) columns, depletion Buffer A, and depletion Buffer B were obtained from Agilent Technologies (Wilmington, DE). Oasis HLB cartridges (1 cm3, 30 mg) were acquired from Waters (Milford, MA). Strata-X 33u Polymeric RP



and Strata-X-C 33u Polymeric SCX cartridges (30 mg/1 mL) were from Phenomenex (Torrance, CA). Immobiline DryStryp pH 3-10 (24 cm), immobilized pH gradient buffer (IPG buffer) pH 310, glycerol 50%, and mineral oil were received from Agilent Technologies.

Sample Preparation The CSF sample used in this work was previously prepared20 and further fractionated with OGE for the purpose of the current study. Ninety-six aliquots of 400 µL of the commercial pooled CSF sample (i.e., in total 38.4 mL of CSF) were evaporated with a vacuum centrifuge (Thermo Scientific). The dried samples were diluted in 125 µL of Buffer A containing 0.00965 mg/mL LACB. A volume of 120 µL was filtered with 0.22 µm filter plate from Millipore. Abundant CSF proteins were removed from the filtered CSF sample solutions (100 µL loaded on column), following the manufacturer instructions with slight modifications, using MARS columns and HPLC systems (Thermo Scientific, San Jose, CA) equipped with an HTC-PAL (CTC Analytics AG, Zwingen, Switzerland) fraction collector. After immunodepletion, samples were snapfrozen and stored. Buffer exchange was performed with Strata-X 33u Polymeric RP (30 mg/1 mL) cartridges mounted on a 96-hole holder and a vacuum manifold, as previously described21. Samples were subsequently evaporated and stored at −80 °C. Dried samples were subjected to reduction, alkylation, digestion, TMT 6-plex labeling, pooling and solid phase extraction (SPE) sample purification (Oasis HLB and SCX) using a 4-channels Microlab Star liquid handler workstation (Hamilton, Bonaduz, Switzerland) in a 96-well-plate format and according to a previously reported protocol21-24. Briefly, each sample was dissolved in 95 µL of TEAB 100 mM and 5 µL of 2% SDS. A volume of 5.3 µL of TCEP (20 mM) was added and incubation was performed for 1 h at 55 °C. A volume of 5.5 µL of IAA 150 mM was added (incubation for 1 h in darkness). Enzymatic digestion was performed via the addition of 10 µL of trypsin/Lys-C at 8 ACS Paragon Plus Environment

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0.25 µg/µL in 100 mM TEAB (incubation overnight at 37 °C). TMT labeling was performed via the addition of 0.8 mg of TMT 6-plex reagent in 41 µL of CH3CN (incubation for 1 h at room temperature). After reaction, a volume of 8 µL of hydroxylamine 5% in H2O was added to each tube to react for 15 min. Samples from a given TMT 6-plex experiment were pooled together in a new tube. Pooled samples were further purified with Oasis HLB, followed by SCX SPE. All samples were resuspended in 200 µL of H2O/CH3CN/FA 96.9/3/0.1; 75 µL of the 16 resulting pooled samples were mixed together (to get enough material), dried, and dissolved in 3232.8 µL H2O with 345.6 µL glycerol 50% and 21.6 µL of IPG buffer pH 3-10. The sample was fractionated with OGE according to a previously published protocol25, using the 3100 OFFGEL Fractionator (Agilent Technologies).

RP-LC MS/MS analysis The twenty-four fractions were dissolved in 50 µL H2O/CH3CN/FA 96.9/3/0.1 for RP-LC MS/MS. RP-LC MS/MS was performed with an Orbitrap Fusion Lumos Tribrid mass spectrometer and an Ultimate 3000 RSLC nano system (Thermo Scientific). Proteolytic peptides (injection of 5 µL of sample) were trapped on an Acclaim PepMap 75 µm × 2 cm (C18, 3 µm, 100 Å) precolumn and separated on an Acclaim PepMap RSLC 75 µm × 50 cm (C18, 2 µm, 100 Å) column (Thermo Scientific) coupled to a stainless steel nanobore emitter (40 mm, OD 1/32 in.). The column was heated to 50 °C using a PRSO-V1 column oven (Sonation, Biberach, Germany). Peptides were separated using the following gradient of solvent A (H2O/CH3CN/FA 97.9/2/0.1) and solvent B (H2O/CH3CN/FA 19.92/80/0.08): from 1% B to 6.3% B in 1 min, from 6.3% B to 11% B in 11 min, from 11% B to 17.5% B in 71 min, from 17.5% B to 25.5% B in 46 min, from 25.5% B to 40% B in 28 min, and maintained at 98% B for 8 min; the column was reconditioned at 6.3% B for 15 min. The flow rate was 220 nL/min with a total analysis time of 9 ACS Paragon Plus Environment


180 min. Data were acquired using a data-dependent method. A positive ion spray voltage of 1500 V and a transfer tube temperature of 275 °C were set up. For MS survey scans in profile mode, the Orbitrap resolution was 120000 at m/z = 200 (automatic gain control target of 2 × 105) with a m/z scan range from 300 to 1500, RF lens set at 30%, and maximum injection time of 100 ms. For MS/MS with higher-energy collisional dissociation (HCD) at 35% of the normalized collision energy, AGC target was set to 1 × 105 (isolation width of 1.2 in the quadrupole), with a resolution of 15000 at m/z = 200, first mass at m/z = 100, and a maximum injection time of 100 ms with Orbitrap acquiring in profile mode. A duty cycle time of 3 s (top speed mode) was used to determine the number of precursor ions to be selected for HCD-based MS/MS. Ions were injected for all available parallelizable time. Dynamic exclusion was set for 60 s within a ± 10 ppm window. A lock mass of m/z = 445.12002 was used.

Data Processing and Analysis Proteome Discoverer (version 1.4, Thermo Scientific) was used as data processing interface. Identification was performed against the human UniProtKB/Swiss-Prot database (2017/07 release) including the LACB sequence (20225 sequences in total). Mascot (version 2.4.2, Matrix Sciences, London, U.K.) was used as search engine. Variable amino acid modifications were oxidized methionine, deamidated asparagine/glutamine, and 6-plex TMT-labeled peptide amino terminus; 6-plex TMT-labeled lysine was set as fixed modifications as well as carbamidomethylation of cysteine. Trypsin was selected as the proteolytic enzyme, with a maximum of two potential missed cleavages. Peptide and fragment ion tolerance were set to10 ppm and 0.02 Da, respectively. All Mascot result files were loaded into Scaffold Q+S 4.8.4 (Proteome Software, Portland, OR) to be further searched with X! Tandem (The GPM, thegpm.org; version CYCLONE (2010.12.01.1)). The false discovery rate (FDR) in Scaffold was 10 ACS Paragon Plus Environment

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calculated with the method of Kall et al.26 by dividing the number of reverse hits by the total number of hits. At the end, the FDRs were 0.9% for proteins, 0.1% for peptides and 0.06% for peptide spectrum matches (that means 29 false positives at each level). The FDR at protein level was based on a target-decoy strategy; this strategy cannot model all types of error. Therefore, not all proteins that passed the threshold are “confidently identified”. GOrilla27 was used for Gene Ontology (GO) term enrichment. The dataset was used initially to generate a spectral library of TMT-labeled peptides for another study. That is why CSF samples were labeled with TMT; this quantitative information was not exploited in the present report.

Detection of missing proteins The list of all identified proteins was searched against the list of 2186 MPs (i.e., protein existence (PE) 2-4) retrieved from neXtProt28 (2018/01/17 release). The uniqueness of all peptide sequences mapping to MPs was checked with the uniqueness checker of neXtProt29. Accordingly to the HPP Guidelines, only unique non-nested peptides with sequence length ≥ 9 amino acids were kept.

Data Availability The MS proteomic data has been deposited to the ProteomeXchange Consortium30 (http:// proteomecentral.proteomexchange.org) via the PRIDE partner repository31 with the dataset identifier PXD009646 (Username: [email protected], Password: 1iOqr37H).

Results and Discussion 11 ACS Paragon Plus Environment


A Deep Human CSF Proteome

Figure 2: Proteomic workflow used for the analysis of the human CSF samples. CSF samples were depleted of 14 abundant proteins; the flow-through proteins were submitted to reduction, alkylation, digestion, and isobaric labeling. The pooled sample was separated in 24 fractions with OGE and each fraction was analyzed with LC MS/MS using a hybrid Orbitrap mass spectrometer (see the Experimental Section).

Our proteomic workflow to analyze human CSF is presented in Figure 2. A commercial pool of “normal” CSF samples was first depleted of 14 proteins known to be abundant in plasma. The flow-through proteins were then subjected to reduction, alkylation, and digestion with trypsin/Lys-C. TMT 6-plex labeling was performed before sample pooling and fractionation with OGE. The resulting 24 fractions were analyzed independently with LC MS/MS using a hybrid Orbitrap mass spectrometer (i.e., Orbitrap Fusion Lumos). The analysis allowed the identification of 20689 peptides mapping on 3379 proteins (peptides and proteins lists are given in Supporting Information Table S1). This represents, to our knowledge, the largest protein set


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from “normal” human CSF reported so far. Our large dataset completes those previously published (see Table 1).

Gene Ontology Term Enrichment Analysis of the CSF Proteome A GO term enrichment analysis was performed on the following ontologies: (a) biological process (BP), (b) molecular function (MF) and (c) cellular component (CC), on the obtained CSF proteome with respect to the full human proteome (Table 2).

Table 2: GO term enrichment of the genes representative of the 3379 proteins identified in “normal” human CSF. GO term enrichment analysis was performed with GOrilla27 on the three ontologies, i.e., (a) BP, (b) MF and (c) CC. The background used for the enrichment analysis was the full human proteome (UniProtKB/Swiss-Prot 2017/07 release; 20224 entries). In the table, only terms with p-value32 under 10-5 and fold enrichment greater than 5, are displayed. (a) Enriched biological process (BP) GO terms GO number

GO term

Number of proteins identified in CSF

GO:0006957 Complement activation, alternative pathway GO:1902285 Semaphorin-plexin signaling pathway involved in neuron projection guidance GO:1902287 Semaphorin-plexin signaling pathway involved in axon guidance GO:0034371 Chylomicron remodeling GO:1902284 Neuron projection extension involved in neuron projection guidance GO:0048846 Axon extension involved in axon guidance GO:0021785 Branchiomotor neuron axon guidance GO:0048842 Positive regulation of axon extension

Total number of Fold protein in human enrichment UniProtKB/SwissProt

13

13

5.6

13

13

5.6

12

12

5.6

9 8

9 8

5.6 5.6

8

8

5.6

7 7

7 7

5.6 5.6



GO:0097119 GO:0010757 GO:2000427 GO:1903551 GO:1905005

GO:0034370 GO:0042340 GO:0071526 GO:0001867

involved in axon guidance Postsynaptic density protein 95 clustering Negative regulation of plasminogen activation Positive regulation of apoptotic cell clearance Regulation of extracellular exosome assembly Regulation of epithelial to mesenchymal transition involved in endocardial cushion formation Triglyceride-rich lipoprotein particle remodeling Keratan sulfate catabolic process Semaphorin-plexin signaling pathway Complement activation, lectin pathway

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6

6

5.6

6

6

5.6

6

6

5.6

6

6

5.6

6

6

5.6

12

13

5.17

11 28 9

12 31 10

5.14 5.06 5.04

Fold enrichment

11 10 8 7 6

Total number of protein in human UniProtKB/SwissProt 11 10 8 7 6

6

6

5.6

10 19

11 21

5.09 5.07

(b) Enriched molecular function (MF) GO terms GO number

GO term

Number of proteins in this study

Semaphorin receptor activity Serine-type carboxypeptidase activity Insulin-like growth factor II binding Axon guidance receptor activity Coreceptor activity involved in Wnt signaling pathway, planar cell polarity pathway GO:0060228 Phosphatidylcholine-sterol Oacyltransferase activator activity GO:0048407 Platelet-derived growth factor binding GO:0045499 Chemorepellent activity GO:0017154 GO:0004185 GO:0031995 GO:0008046 GO:1904929

5.6 5.6 5.6 5.6 5.6

(c) Enriched cellular component (CC) GO terms GO number

GO:0042627 GO:0002116 GO:0001527 GO:0005579

GO term

Number of proteins in this study

Chylomicron Semaphorin receptor complex Microfibril Membrane attack complex

13 11 10 7 14

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Total number of protein in human UniProtKB/SwissProt 13 11 10 7

Fold enrichment

5.6 5.6 5.6 5.6

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GO:0071682 Endocytic vesicle lumen GO:0005614 Interstitial matrix GO:0005583 Fibrillar collagen trimer

17 11 11

18 12 12

5.29 5.14 5.14

For the three GO categories, terms related to semaphorin were enriched, i.e., “semaphorin-plexin signaling pathway involved in neuron projection guidance” (13 genes out of 13), “semaphorinplexin signaling pathway involved in axon guidance” (12 genes out of 12), “semaphorin-plexin signaling pathway” (28 genes out of 31), “semaphorin receptor activity” (11 genes out of 11), and “semaphorin receptor complex” (11 genes out of 11). Semaphorin/neuropilin/plexin complexes are involved in the development of neuronal connectivity by regulating axon guidance33. As a matter of fact, the GO terms “neuron projection extension involved in neuron projection guidance” (8 genes out of 8), “branchiomotor neuron axon guidance” (7 genes out of 7), “axon extension involved in axon guidance” (8 genes out of 8), “positive regulation of axon extension involved in axon guidance” (7 genes out of 7), “axon guidance receptor activity” (7 genes out of 7)

and “chemorepellent activity” (19 genes out of 21) were also enriched.

Semaphorins and plexins are potential prognostic biomarkers and therapeutic targets in glioma pathogenesis34 and in multiple sclerosis35-36. The term “postsynaptic density protein 95 clustering” was also significantly enriched (6 genes out of 6) (Table 2a). This process is important for the development of excitatory synapses in the brain. Among the other enriched GO terms in Table 2 were terms linked to innate immunity, i.e., “complement activation, alternative pathway” (13 genes out of 13), “complement activation, lectin pathway” (9 genes out of 10), “negative regulation of plasminogen activation” (6 genes out of 6), “positive regulation of apoptotic cell clearance” (6 genes out of 6), and “membrane attack complex” (7 genes out of 7). Proteins from the coagulation and complement cascades have



already been detected and studied in the CSF; they have been proposed as markers for various neurological diseases such as multiple sclerosis or AD37-38. Other enriched GO terms in Table 2, such as “chylomicron remodeling” (9 genes out of 9), “triglyceride-rich lipoprotein particle remodeling” (12 genes out of 13) and “chylomicron” (13 genes out of 13) are linked to the apolipoprotein family. The 15 apolipoproteins A-II, C-I, L, AV, B receptor, B-100, C-III, A-IV, C-II, E (APOE), M, D, A-I, apolipoprotein (a), and beta-2glycoprotein 1 were identified here in CSF. Concentration measurement of these proteins is clinically relevant39; for instance, APOE is a biomarker related to AD but it is not the only apolipoprotein biomarker of the pathology. A recent publication showed a variation of CSF APOA1 levels in AD patient40. The term “insulin-like growth factor II binding” was also enriched in the CSF dataset (8 genes out 8). It was shown that CSF levels of insulin-like growth factor II and some of its binding proteins were altered in patients with AD and might also serve as AD indicators41. In general, the GO term enrichment analysis clearly indicated that the CSF proteome is enriched in proteins known to be involved in brain development and/or diseases. According to The Human Protein Atlas42, 74% of all human proteins are expressed in the brain. Overall, 1460 of these genes show an elevated expression in the brain compared to other tissue types43. Interestingly, 500 out of these 1460 proteins (i.e., 34%) were present in our CSF dataset, including those corresponding to the top three over-expressed genes: OPALIN, GFAP and OMG. The list of these 500 proteins is given in Supporting Information Table S2. Among those proteins, some principal biomarkers of several neurodegenerative diseases were present, such as microtubule-associated tau protein (MAPT) and neurogranin (NRGN) for AD, and alphasynuclein (SNCA) for PD44. CSF is a complex body fluid in which 20-30% of proteins are


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thought to be derived from the brain; both CSF proteome and brain-specific proteome overlap to a certain extent only. It is therefore not surprising not to identify directly in CSF the full set of proteins highly expressed in the brain.

Coverage Comparison with Other CSF Proteome Datasets Other in-depth proteomic studies of CSF were reported in the past, using different sample preparation methods (see Table 1). In the study by Guldbrandsen et al.11, three different sample preparations (i.e., SDS-PAGE, mixed mode RP-anion exchange and hydrazide-based glycopeptide capture) were used and CSF samples were analyzed with an LTQ-Orbitrap Velos Pro to obtain a final protein set of 3081 entries. Data were searched using X! Tandem and OMSSA and 1% FDR at protein, peptide and peptide spectrum match was applied. Zhang et al.2, from three CSF samples (depleted and not depleted CSF samples separated in 30 fractions with high-pH RP-LC) analyzed with a Triple TOF 5600 mass spectrometer, identified 3256 proteins. In a more recent report, Kroksveen et al.12 analyzed six pools of CSF (from patients with multiple sclerosis and controls) fractionated in a total of 115 fractions, enriched or not in glycoproteins; the authors identified 3006 proteins in CSF by searching with X! Tandem, MyriMatch and Comet and applying a 1% FDR at both protein and peptide levels.



Figure 3: Venn diagram comparing the human CSF proteome of in-depth proteomic studies. The CSF proteome of the present study is compared with two other CSF proteomic studies by Kroksveen et al.12 and Guldbrandsen et al.11. Each of the study independently identifies around 3000 proteins with their respective specificities (e.g., sample preparation or mass spectrometer used). Around 40% of all proteins are common to all three datasets; even if a plateau seems to be reached in terms of number of CSF proteins identified using current technologies, by merging these three datasets, a consequent CSF proteome of 4814 proteins is obtained.

In Figure 3, we compared the proteins identified in CSF by Kroksveen et al., Guldbrandsen et al. and us in the present study (the study by Zhang et al. was not considered here as the protein list with one specific peptide criterion was not available from this work (see Method Section)). From the Venn diagram, 1913 proteins (i.e., 39.7%) were common to the three studies; principal 18 ACS Paragon Plus Environment

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AD biomarkers (i.e., tau protein, amyloid-β and APOE) and thirteen apolipoproteins were commonly identified. Putting a central focus on the proteins included in this redundant and confident CSF proteome appears extremely relevant for clinical studies and biomarker discoveries; these proteins are certainly identifiable in CSF, independently to the sample preparation workflow or the MS instrumentation used.

Contribution of Sample Fractionation and MS Technology to the Characterization of the CSF Proteome Thanks to the recent development of MS technologies in proteomics, it is now possible to exceed drastically the coverage of identified proteins in CSF (Figure 4). In one of our previous study (referred as Study 1 in Figure 4), we analyzed the same CSF sample as studied herein, in 16 replicates, but without fractionation, and injected the samples in triplicates for detection with an LTQ-Orbitrap Elite (see Núñez Galindo et al.23 for more information). In another more recent work (referred as Study 2 in Figure 4; see Macron et al.20 for more information), we analyzed the exact same sample as studied herein (i.e., including OGE for sample fractionation) using an LTQ-Orbitrap Elite. Comparing these studies with the current work (Figure 4), we could assess the respective contribution of i) CSF sample fractionation with OGE and ii) MS technology improvement from LTQ-Orbitrap Elite to Orbitrap Fusion Lumos. In terms of protein identification, the fractionation of the CSF sample with OGE increased the number of proteins by 2.2 fold (i.e., 1000 identified proteins in Study 1 versus 2281 in Study 2). In total, 88 proteins in Study 1 were not identified in Study 2 (i.e., 8.8%). The use of the latest Orbitrap technology increased the number of identified CSF proteins in the present study by 1.5 fold with respect to Study 2 (i.e., 2281 identified proteins in Study 2 versus 3379 in this study). About 89% of the



proteins identified in Study 2 (Figure 4), were also identified herein. The combined two technological factors (i.e., use of fractionation and latest MS technology) allowed multiplying by more than three the number of identified CSF proteins with respect to our initial Study 1.


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Figure 4: Comparison of the proteomic analysis of an identical CSF sample using different sample preparations and mass spectrometry analyses. (a) Representation of the different MSbased proteomic workflows used in Study 1 (Núñez Galindo et al.23), Study 2 (Macron et al.20), and the present study with the number of proteins and peptides identified in CSF for each study. (b) Venn diagram comparing the list of identified proteins in these three studies.

Missing Proteins As its proteome still needs to be characterized fully, human CSF appears as a relevant sample for the validation of the existence of proteins not detected in other body fluids and tissues. MS-based identification of MPs predicted by genomic or transcriptomic analyses is a key goal of the CHPP19. In the last release of neXtProt28 (i.e., 2018/01/17), 2186 proteins were still marked as missing. These proteins are annotated with a “protein existence” (PE) score of 2 when they are predicted from transcriptomic analysis, 3 when they are predicted from genomic analysis and have a homologue in distant species, and 4 when they are only predicted from genomic analysis in human. The status “PE5” defines an uncertain protein. Switzerland contributes to the C-HPP initiative by focusing on the identification and characterization of proteins encoded on chromosome 2. This chromosome counts 1271 proteins of which 98 are still missing (PE2-PE4) and 16 remain uncertain (PE5). One key goal of the present study was to investigate the presence of MPs in a deeply characterized “normal” CSF sample. Nine proteins in our CSF dataset matched with PE2 MPs in accordance to the HPP guidelines45. All nine proteins and their respective peptides are presented in Table 3 (tandem mass spectra are given in Supporting Information I). As recommended by the HPP guidelines, for further confirmation, we synthetized the peptides allowing the identification of an MP in our dataset.



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The synthetic peptides were labeled with TMT and analyzed with MS. All spectra are presented in Supporting Information I. For all peptides (except VVFGIFANIL for which we did not obtain any spectra), we obtained a good match between the endogenous and synthetic peptide tandem mass spectra (Supporting Information I); this strengthen our MP identifications.

Table 3: List of unique peptides of nine or more amino acids allowing the validation of PE2 proteins.

Entry

Protein name

Unique Peptide sequence

Q9P2K9

Protein dispatched homolog 3

DLADFTSETLQR

Chromosom e 1p36.22

Q9P2K9


DTSAAQKPTANR

1p36.22

Q9P2K9


ETPPLEDLAANQSEDPR

1p36.22

Q4G0M1

Erythroferrone

AGPAARPPEPTAER

2q37.3

Q4G0M1 Q9H1Z8

Erythroferrone Augurin

EPPPGNELPR AKEFLGSLK*

2q37.3 2q12.2

Q9H1Z8

Augurin

EAPVPTKTK*

2q12.2

Q9H1Z8

Augurin

HGASVNYDDY

2q12.2

Q9H1Z8

Augurin

HYDEDSAIGPR*

2q12.2

Q9H1Z8

Augurin

TKVAVDENK*

2q12.2

Q9H1Z8 B5MCY 1 B5MCY 1 O60330

Augurin

VAVDENKAK*

2q12.2

Tudor domain-containing protein 15

AAVLTQVSK

2p24.1

Tudor domain-containing protein 15

VVFGIFANIL

2p24.1

Protocadherin gamma-A12

AAHHLVLTASDGGDPVR

5q31.3

O60330


DINDNAPYFR

5q31.3

O60330


YSVPEELEK

5q31.3

Q9Y5E5

Protocadherin beta-4

SYHVEIEATDGGGLSGK

5q31.3

Q9Y5E5

Protocadherin beta-4

VLSDDDKQR

5q31.3

Q5BIV9

Shadow of prion protein

10q26.3

Q5BIV9


Q5BIV9


GLEDEEDGVPGGNGTGPGIYSYR VAAAGAAAGAAAGAAAGLAAGSGWR * YGAPGSSLR*

10q26.3

Q8N6Y1

Protocadherin-20

LYATDADSEER

13q21.2

Q8N6Y1

Protocadherin-20 Multiple epidermal growth factorlike domains protein 11 Multiple epidermal growth factorlike domains protein 11

SAGRPDPQSQLPER

13q21.2

CEELCAPGTHGK

15q22.31

GCQLPCQCR

15q22.31

A6BM72 A6BM72


10q26.3

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A6BM72

Multiple epidermal growth factorlike domains protein 11

ISPALGAER*

15q22.31

*peptide also previously reported in Macron et al.20

Three protocadherins were identified: protocadherin gamma-A12, protocadherin beta-4 and protocadherin-20. Protocadherins are a large family of type I transmembrane proteins that mediate a variety of developmental brain processes including neuronal survival, synaptic maintenance and neuronal circuit formation. Protocadherin gamma-A12 and protocadherin beta4 genes belong to the protocadherin gamma and beta clusters on chromosome 5q31, respectively. Protocadherins from the different clusters are stochastically and combinatorically expressed in neurons, thereby providing an important mechanism to generate neuronal identity46. In mice, gamma-protocadherins have been shown to be expressed by choroid plexus epithelial cells, the primary site of CSF production47. Protocadherin-20 is a non-clustered protocadherin that was initially proposed to be a tumor suppressor48 and was more recently shown to be involved in cortical development49. Its levels were shown to change significantly in multiple sclerosis (Kroksveen et al.12). Another type 1 transmembrane protein, encoded by MEGF11, was unambiguously validated with

three

unique,

non-nested

peptides,

complementing

the

unique

peptide

CDCHNGGQCSPTTGACECEPGYK previously identified in CSF by Guldbrandsen et al.11. One of these peptides, i.e., ISPALGAER, was also observed in our previous CSF analysis using an LTQ-Orbitrap Elite (see Study 2 in Figure 4 and Macron et al.20 for more information). MEGF11 underwent adaptive molecular evolution in primate lineages and might be involved in primate-specific traits50. The prion-like protein shadoo, which was shown to be associated to the outer leaflet of plasma membranes through a glycosylphosphatidylinositol anchor51, was also identified in Macron et



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al.20 which give even more confidence in the identification reported here (Table 3). This protein was clearly identified, with three unique, non-nested peptides; one among them (i.e., VAAAGAAAGAAAGAAAGLAAGSGWR) was previously identified in CSF by Guldbrandsen et al.11. Taken together, these three peptides cover 57 amino acids out of the 101 amino acids that constitute the mature protein after signal sequence and propeptide processing. Although shadoo has

been

found

to

be

glycosylated

in

rodents52,

the

unglycosylated

peptide

GLEDEEDGVPGGNGTGPGIYSYR was detected, suggesting that shadoo glycosylation may be incomplete in human. Two secreted proteins encoded on chromosome 2 were identified: erythroferrone and augurin. Erythroferrone is a recently discovered hormone linking erythropoiesis and iron metabolism, and is required for the rapid compensatory response to hemorrhage53. As the prion-like protein shadoo, augurin was also identified in Macron et al.20. This hormone-like protein, mostly expressed in the choroid plexus, has been suggested to be involved in CSF homeostasis and brain injury54-55. As early of 2012, augurin was one of the 104 proteins identified in CSF with LCMALDI MS56. In our dataset, it was identified with six unique peptides, among which, one (i.e., HYDEDSAIGPR) was previously reported in pituitary gland57 and cardia58. Several studies reported that augurin can undergo a complex maturation process, depending on the cell type, which might explain its pleiotropic function in injury, inflammation, infection, ageing and malignancy59. The C-terminal peptide (133-148) was shown to interact with TLR460 and the mature form (71-132) was shown to bind to multiple scavenger receptors involved in innate immunity61. The peptides identified in our proteomic analysis of CSF cover the sequences of the reported propeptides (Figure 5), indicating that the protein does exist in its long form in the CSF and confirming results of previous western-blot experiments55.


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Figure 5: Representation of the sequence of augurin. The representation of this small protein shows a signal peptide of 31 amino acids followed by a first 37 amino acid propeptide, then the mature protein (64 amino acids) and finishing by a short propeptide of only 16 amino acids. In yellow is represented the extent of sequence that we here covered in CSF with MS.

Protein dispatched homolog 3 (DISP3) was identified with three peptides, among which one (i.e., ETPPLEDLAANQSEDPR) was already observed in CSF by Guldbrandsen et al.11. It is a sterolsensing domain-containing protein, highly expressed in the nervous system and involved in neuron differentiation62-63. Tudor domain-containing protein 15 (TDRD15) is an uncharacterized protein encoded on chromosome 2. It is the only one from the 28 Tudor domain-containing proteins that had not been previously validated. Its identification in CSF is surprising since TDRD15 mRNA expression seems limited to testis according to The Human Protein Atlas, and its closest paralogs TDRD6 and TDRD1 are both involved in spermatogenesis64. However, Tudor domains are known to be involved in the recognition of methylated arginines65, and it was shown that human CSF contains relatively high endogenous protein methylase activity66. The tandem mass spectrum of VVFGIFANIL peptide identifying B5MCY1 was not of good quality and unfortunately, its synthetic peptide was not detected with LC MS/MS. Therefore, we are not confident about this identification and proposing the protein validation. Even if this protein was



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identified with another peptide (AAVLTQYSK), according to HPP guidelines it is not enough for the validation of this MP. In the dataset, we also identified putative protein shisa-8, a protein classified as uncertain in UniProtKB/Swiss-Prot and neXtProt. The SHISA8 gene has orthologs in other vertebrates, and is considered as a protein-coding gene by other databases such as HUGO Gene Nomenclature Committee. The peptide CSNYDTPAWVQTGRPPA identified herein was already part of the human CSF built from PeptideAtlas; the peptide RAGAPEAQGPAAPGTTAPEGGDR was also previously identified but with the two first amino acids cleaved. Based on these observations, the status of SHISA8 entry in UniProtKB/Swiss-Prot and neXtProt is currently under review (Table 4 and Supplementary Information II).

Table 4: List of unique peptides of nine or more amino acids allowing the validation of SHISA8. Entry

Protein Name

Unique Peptide Sequence

Chromosome

B8ZZ34

Putative protein shisa-8

CSNYDTPAWVQTGRPPA

22q13.2

B8ZZ34


RAGAPEAQGPAAPGTTAPEGGDR

22q13.2

B8ZZ34


VAPPGLAAAAAAR

22q13.2

Seventeen proteins of the MP list were only detected with one unique peptide in our CSF dataset respecting the guidelines size criterion (the list is available in Supporting Information Table S3). However according to PeptideAtlas67, for four of them (Table 5 and Supplementary Information III), another unique peptide with a sequence length ≥ 9 amino acids has previously been reported in other studies. Their validation as PE1 proteins should therefore also be taken into consideration. These four proteins are four-jointed box protein 1 (FJX1), transmembrane protein 178A (TMEM178A), transmembrane protein 74B (TMEM74B) and RING finger protein


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112 (RNF112). Of note, peptides mapping on FJX1 and TMEM178A were also detected in our previous CSF analysis using an LTQ-Orbitrap Elite (see Study 2 in Figure 5 and Macron et al.20 for more information). For the four peptides allowing the identification of these MPs, a synthetic analogue was purchased, labeled with TMT and analyzed with MS. Tandem mass spectra are presented in Supporting Information III. For all of them, fragmentation patterns of endogenous and synthetic peptides correlated.

Table 5: List of unique peptides of nine or more amino acids allowing the validation of PE2 proteins thanks to the contribution of this study and previous works reporting other unique peptides (referred here as additional peptides).

Entry

Protein Name

Unique Peptide Sequence

Chromosome

Q8NBL3

Transmembrane protein 178A

AGADPPDQK*

2p22.1

Additional peptide Q86VR8

TIQQDEWHLLHLR Four-jointed box protein 1

Additional peptides

GLEEQVPPGFSEAQAAAWLEAAR

11p13

TELPASRPPEDR* GAQWAQVQEELR

Q9ULX5

RING finger protein 112

Additional peptide Q9NUR3 Additional peptide

LLEGDREPLLQEE

17p11.2

SFLLNHLLQGLPGLESGEGGRPR Transmembrane protein 74B

SAPLGPVAPTR

20p13

LGSSPSPPGGVSSLPR

*peptides also previously reported in Macron et al.20 Four-jointed box protein 1 is a secreted protein that regulates dendrite extension68. An additional peptide, TELPASRPPEDR, was identified in one of our other work20. Another unique peptide (FPLPPPLAWDAR) of four-jointed box protein 1 was identified in CSF by another group according to the CSF-PR-2.0 tool69. Transmembrane protein 178A is encoded by chromosome 2 and was shown to act as a negative regulator of osteoclast differentiation70. According to The Human Protein Atlas, it is highly expressed in the brain, where it might play an active role as 27 ACS Paragon Plus Environment


well.

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RING finger protein 112, also called neurolastin, is also a transmembrane protein

abundantly expressed in the brain. In mice, it has been shown to be a GTPase from the dynamin family that plays a crucial role in synaptic transmission71-72. Transmembrane protein 74B is a broadly expressed uncharacterized transmembrane phosphoprotein. Its paralog TMEM74 plays an essential role in autophagy73. Taken all together, we anticipate that the present data will be used to validate the existence of 12 MPs (i.e., 8 directly from this study (Table 3) (having removed Tudor domain-containing protein 15; see above) and 4 via the contributions of this study and others (Table 5) in neXtProt including three of chromosome 2.

Uncharacterized PE1 Proteins Using the SPARQL query provided as use example 22 from the neXtProt27 advanced query tool (www.nextprot.org/proteins/search?mode=advanced&queryId=NXQ_00022),

we

determined

that 1260 out of the 17470 PE1 proteins have no function annotated in 2018/01/17 release. This query retrieves proteins with no function annotated, either experimentally determined or inferred by similarity to characterized proteins, as well as proteins annotated with broad GO terms that reflect their general ability to bind proteins or ions but are not linked to any function (GO:0005509, calcium ion binding; GO:0008270, zinc ion binding; GO:0005515, protein binding, GO:0042802, identical protein binding; GO:0051260, protein homooligomerization; GO:0005524, ATP-binding, GO:0000287 magnesium-binding, GO:0003676 nucleic acid binding, GO:0003824 catalytic activity, GO:0007165 signal transduction, GO:0035556 intracellular signal transduction). The C-HPP recently launched an experimental characterization effort on these unannotated PE1 proteins (uPE1). Interestingly, 116 uPE1 proteins were detected


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in our dataset (Supporting Information Table S4). Three of them had not been identified with MS before; instead, their existence had been validated using techniques such as structural analysis

(LRRC3B)

or

protein-protein

interaction

analysis

(T-ENOL,

EXOC3-AS1).

Unexpectedly, mammaglobin-A (SCGB2A2),74 was identified. This protein is a marker of breast cancer and has been found in the CSF of patients with brain metastasis75. Two identified uPE1 proteins are known to be encoded by genes on which mutations can cause neurological diseases (TMEM185A in FRAXF syndrome and MIPOL1 in Mirror-image polydactyly). Two others are involved in autoimmune diseases: anti-IgLON5 antibodies lead to a characteristic tauopathy with sleep disorder76 and anti-SBSN antibodies may contribute to the pathogenesis of neuropsychiatric systemic lupus erythematosus77. Two proteins from the list (Supporting Information Table S4) have been subjected to dedicated studies which failed to determine their function: the oligodendrocyte marker opalin (OPALIN) 78 was thought to be involved in myelin formation, but knock-out mice do not display any myelin-related phenotype. Similarly, synapse-associated protein 1 (SYAP1)79 is highly expressed in the CNS but knock-out mice do not display any brain-related phenotype. Two other proteins underwent preliminary functional analysis: transmembrane 9 superfamily member 3 (TM9SF3)80-81, a Golgi apparatus protein that might be involved in cell proliferation, and protein FAM107B82 which may be involved in corticogenesis. Eight proteins have been characterized recently. Requests for functional annotation updates have been sent to UniProtKB/Swiss-Prot curators. ER membrane protein complex subunit 10 (EMC10)83 is an angiogenic growth factor promoting tissue repair after myocardial infarction. Protein FAM19A584-85 is a chemokine which may be involved in osteoclast formation and may regulate vascular pathology upon injury. Reticulocalbin-3 (RCN3)86 is an endoplasmic reticulum



lumen protein involved in lung maturation. Zinc finger SWIM domain-containing protein 6 (ZSWIM6)87-88 is involved in neuronal development in mouse and human, and mutations on this gene have been found to cause acromelic frontonasal dysostosis and severe intellectual disability. Immunoglobulin superfamily member 21 (IgSF21)89 was shown to promote the differentiation of inhibitory synapses in the brain. Breast carcinoma-amplified sequence 1 (BCAS1)90 is expressed in a subset of oligodendrocytes and may be involved in myelin formation. Interferon alphainducible protein 27-like protein 2 (IFI27L2)91, also called ISG12B, is a mitochondrial protein involved in apoptosis. Vacuolar protein sorting-associated protein 13D (VPS13D) 92-93 is another mitochondrial protein that regulates mitochondrial size and clearance, and mutations on its gene were found to cause childhood-onset movement disorders. Taken together, our careful literature mining showed that 18 uPE1 proteins identified in this study are either already characterized or will be characterized soon due to their known associations with diseases, while 2 PE1 proteins in CSF were described as difficult to functionally characterize.

The remaining 96 proteins, including six encoded on chromosome 2 would be interesting to characterize further, using appropriate experimental approaches. According to neXtProt, 48 of them are associated or integral to membranes, sixteen are secreted, six are cytoplasmic and/or nuclear, and two are mitochondrial. Their presence in CSF suggests a possible brain-related function.

Conclusion


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We present in this study a deep characterization of the “normal” human CSF proteome encompassing 3379 proteins and 20689 peptides. The list of CSF proteins contains biomarkers of neurodegenerative diseases such as AD or PD. We consider such a resource as a valuable starting point for further clinical investigations and the development of targeted assays for specific proteins involved in various pathologies of the CNS. Importantly, our deep in-depth unbiased proteomic profiling of a single pooled sample of human CSF detected 26 MPs (with at least one unique peptide of nine or more amino acids); it should allow the validation of 12 MPs in the context of the C-HPP.

Supporting Information Supporting Information I. Annotated spectra for all unique peptides of more than nine amino acids allowing the validation of PE2 proteins and their respective synthetic peptide tandem mass spectra. Supporting Information II. Annotated spectra for all unique peptides of more than nine amino acids allowing the validation of B8ZZ34. Supporting Information III. Annotated spectra for all unique peptides of more than nine amino acids allowing the validation of PE2 proteins thanks to the contribution to this study and peptides already reported in other studies present in PeptideAtlas and their respective synthetic tandem mass spectra. Table S1. List of peptides and proteins identified in this dataset.



Table S2. List of proteins from this dataset over-expressed in the brain according to The Human Protein Atlas. Table S3. List of missing proteins identified with only one unique peptide with more than 9 amino acids. Table S4. List of uPE1 proteins identified.

Corresponding Authors Nestlé Institute of Health Sciences, EPFL Innovation Park, Bâtiment H, 1015 Lausanne, Switzerland; Email: [email protected], Phone: +41 21 632 6114, Fax: +41 21 632 6499

Notes The authors declare no competing financial interest.

Abbreviations AD

Alzheimer Disease

BP

Biological Process

CC

Cellular Component

CNS

Central Nervous System

CSF

CerebroSpinal Fluid

FDR

False Discovery Rate

FT-ICR

Fourier Transform Ion Cyclotron Resonance 32 ACS Paragon Plus Environment

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GO

Gene Ontology

HCD

Higher-energy Collision Dissociation

HPP

Human Project Proteome

IT

Ion Trap

LACB

β-lactoglobulin

LC

Liquid Chromatography

LTQ

Linear Trap Quadrupole

MALDI-TOF

Matrix-Assisted Laser Desorption Ionization-Time Of Flight

MF

Molecular Function

MP

Missing Protein

MS

Mass Spectrometry

MS/MS

Tandem Mass Spectrometry

OGE

Off-Gel Electrophoresis

PD

Parkinson Disease

PE

Protein Existence

Q

Quadrupole

RP

Reversed-Phase

SCX

Strong Cation-Exchange



SDS-PAGE

Sodium Dodecyl Sulphate Polyacrylamide-Gel Electrophoresis

SPE

Solid Phase Extraction

TMT

Tandem Mass Tag

uPE1

Unannotated protein with Protein Existence 1 level

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Deep Dive on the Proteome of Human Cerebrospinal Fluid: A

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