Enhanced Detection of CNS Cell Secretome in Plasma Protein-Depleted Cerebrospinal Fluid Eric Thouvenot,†,‡,§,|,⊥ Serge Urbach,†,‡,§,| Christelle Dantec,†,‡,§,| Joe¨l Poncet,†,‡,§,| Martial Se´veno,†,‡,§,| Edith Demettre,†,‡,§,| Patrick Jouin,†,‡,§,| Jacques Touchon,⊥ Joe¨l Bockaert,†,‡,§,| and Philippe Marin*,†,‡,§,| Centre National de la Recherche Scientifique, UMR 5203, Institut de Ge´nomique Fonctionnelle, Montpellier, France, Institut National de la Sante´ et de la Recherche Me´dicale, U661, Montpellier, F-34094 France, Universite´ Montpellier 1, Montpellier, F-34094 France, Universite´ Montpellier 2, Montpellier, F-34094 France, and Service de Neurologie, Centre Hospitalier Universitaire de Montpellier, Montpellier, F-34295 France Received May 27, 2008
Human cerebrospinal fluid (CSF) proteome is actively investigated to identify relevant biomarkers and therapeutic targets for neurological disorders. Approximately 80% of CSF proteome originate from plasma, yielding a high dynamic range in CSF protein concentration and precluding identification of potential biomarkers originating from CNS cells. Here, we have adapted the most complete multiaffinity depletion method available to remove 20 abundant plasma proteins from a CSF pool originating from patients with various cognitive disorders. We identified 622 unique CSF proteins in immunodepleted plus retained fractions versus 299 in native CSF, including 22 proteins hitherto not identified in CSF. Parallel analysis of neuronal secretome identified 34 major proteins secreted by cultured cortical neurons (cell adhesion molecules, proteins involved in neurite outgrowth and axonal guidance, modulators of synaptic transmission, proteases and protease inhibitors) of which 76% were detected with a high confidence in immunodepleted CSF versus 50% in native CSF. Moreover, a majority of proteins previously identified as secretory products of choroid plexus cells or astrocytes were detected in immunodepleted CSF. Hence, removal of 20 major plasma proteins from CSF improves detection of brain cell-derived proteins in CSF and should facilitate identification of relevant biomarkers in CSF proteome profiling analyses. Keywords: cerebrospinal fluid • immunodepletion • neuron • secretome
1. Introduction Cerebrospinal fluid (CSF) is mainly produced by choroid plexuses (CPs) located in cerebral ventricles. It circulates through the ventricular system and around the brain and the spinal cord within the subarachnoid space. CSF is a sample of choice in the research of biomarkers for neurological disorders.1,2 CSF samples are easily accessible by standard lumbar puncture techniques. Moreover, CSF is in direct contact with brain interstitial fluid and biochemical changes in CSF reflect pathological alteration of CNS physiology. Accordingly, CSF receives proteins secreted by different CNS cell populations (secretome), especially choroid plexus epithelial cells (CECs), glial cells and neurons. Therefore, any change in CSF protein composition may be indicative of altered brain protein expression and secretion in CNS disorders. * To whom correspondence should be addressed. Institut de Ge´nomique Fonctionnelle, 141 rue de la Cardonille, 34094 Montpellier Cedex 5, France. Phone, +33 467 14 29 83; fax, +33 4 67 54 24 32; e-mail, philippe.marin@ igf.cnrs.fr. † Institut de Ge´nomique Fonctionnelle. ‡ Institut National de la Sante´ et de la Recherche Me´dicale. § Universite´ Montpellier 1. | Universite´ Montpellier 2. ⊥ Centre Hospitalier Universitaire de Montpellier. 10.1021/pr8003858 CCC: $40.75
2008 American Chemical Society
Despite tremendous efforts to decipher CSF proteome during the past few years, a number of proteins identified as secretion products of CNS cells have never been detected in CSF, due to their low concentration. One major reason is certainly the masking effect of plasma proteins, which are recovered in CSF by active transport via pinocytosis across the blood-brain barrier and represent about 80% of total CSF proteome.3 Different methods prompted by plasma proteome studies have been used to remove the most abundant plasma-derived proteins from CSF, leading to increased sensitivity of CSF protein identification. A first strategy based on multiple precipitation steps, mainly removed albumin and immunoglobulins (Igs) and allowed better analyses, but was rather unspecific and eliminated a number of proteins exhibiting a low expression level.1,4,5 Alternatively, immunodepletion of the major plasma proteins, either solely albumin,6,7 albumin and Igs,7 or the 6 major plasma proteins 8-10 reduced the dynamic range of CSF proteome and enhanced identification rates. These CSF fractionation methods, coupled with high sensitive mass spectrometry technologies, have provided extensive maps of human CSF proteome,11 but they only slightly improved the sensitivity of analysis compared to analysis of native CSF.10 An alternative approach based on general depletion of highJournal of Proteome Research 2008, 7, 4409–4421 4409 Published on Web 09/06/2008
research articles abundance CSF proteins using a hexapeptide combinatorial library immobilized on beads also allowed identification of a greater number of proteins in depleted versus native samples.12 Here, we have adapted the most complete multiaffinity depletion method available (ProteoPrep 20) to remove 20 of the most abundant plasma proteins from a CSF pool of patients with different cognitive disorders, prior to proteomic analysis. This approach allowed identification of a larger number of CSF proteins by LC-MS/MS in fractionated CSF (immunodepleted plus retained fractions), compared to native CSF (622 and 299 nonredundant proteins, respectively), including 22 hitherto not identified proteins. To evaluate the sensitivity of this approach toward brain-derived proteins, that is, potential biomarkers of CNS diseases, we have established for the first time a proteomic map of major proteins secreted by cultured cortical neurons (secretome). This includes cell adhesion molecules, proteins involved in neurite outgrowth and axonal guidance, modulators of synaptic transmission, proteases and protease inhibitors. Comparing fractionated and native CSF proteome with neuronal secretome and already published secretome maps of other CNS cell populations (CECs and astrocytes)13,14 reveals that the depletion method used herein, which removes a large fraction of plasma proteins from CSF, yields a more accurate picture of CSF proteins actually released by CNS cells.
2. Materials and Methods 2.1. CSF Samples. CSF samples were obtained by lumbar puncture of 25 patients undergoing clinical evaluation for cognitive impairment at the Montpellier University Hospital and enrolled in an ongoing biomarker study. The study protocol was approved by the “Sud-Me´diterrane´e IV” Ethics Committee (Montpellier University Hospital) and we obtained written and verbal informed consent from participants at enrollment. Samples were collected in polypropylene tubes and centrifuged at 1500g for 10 min at 4 °C and then at 38 000g for 25 min to remove cells and cell debris, respectively, and stored at -80 °C until use. For each experiment, a 0.2 mL aliquot from each patient was thawed on ice and the 25 samples were mixed to obtain the “CSF pool” used in this study. CSF pool aliquots (2 mL) were ultrafiltered using Centricon Ultracell YM-3 membranes (3 kDa cutoff, Millipore, Bedford, MA) at 7000g for 2 h at 4 °C. Concentrated samples were adjusted to a final protein concentration of 4 mg/mL. 2.2. Multiaffinity Immunodepletion. Concentrated samples were immunodepleted of 20 of the major plasma proteins using the ProteoPrep 20 plasma protein immunodepletion kit (Sigma, St. Louis, MO). This kit is based on an immunodepletion column consisting of a resin bioconjugated with polyclonal IgG antibodies against 20 high-abundance plasma proteins (albumin, apolipoproteins A1, A2 and B, alpha-1-acid-glycoprotein, alpha-1-antitrypsin, alpha-2-macroglobulin, ceruloplasmin, complements C1q, C3 and C4, haptoglobin, fibrinogen, IgAs, IgDs, IgGs, IgMs, plasminogen, transferrin and transthyretin (TTR)). Briefly, ProteoPrep 20 columns were washed with 4 mL of equilibration buffer provided in the kit (EB), spun at 1000g for 1 min and placed on a 1.5 mL collector tube for immediate use. A concentrated CSF aliquot (100 µL) was loaded on the column for 20 min and the column was spun at 1000g for 1 min. The same steps were repeated with another 100 µL CSF aliquot. The flow-through fraction containing immunodepleted CSF (200 µL) was harvested in the collector tube. The column was washed with 4 mL of EB and proteins retained by immunoaffinity (retained fraction) were eluted off with 2 mL 4410
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Thouvenot et al. of Elution Buffer provided in the kit and neutralized with 100 µL of Tris (1 M, pH 8.8). The column was washed with 4 mL of EB and the flow-through fraction was passed again over the column for a second immunodepletion step in order to remove residual plasma proteins not retained during the first chromatography. The resulting flow-through fraction (immunodepleted fraction) was used for proteomic analyses. Protein concentration in native, immunodepleted, and retained fractions was determined with the bicinchoninic acid assay.15 Concentrated CSF samples originating from the same CSF pool were also immunodepleted of 6 major plasma proteins (albumin, IgAs, IgGs, transferrin, alpha-1-antitrypsin and haptoglobin) using the Multiple Affinity Removal System (MARS 6, spin cartridges, Agilent Technologies, Santa Clara, CA). We used the same procedure as in the ProteoPrep 20 immunodepletion method (two immunodepletion cycles) except that 200 µL of concentrated CSF were directly loaded on the spin column. 2.3. Primary Cultures of Cortical Neurons. Primary cultures of cortical neurons were prepared as previously described.16 Briefly, cells dissociated from the cerebral cortex of 16.5 dayold Swiss mouse embryos were plated in serum-free medium on 100 mm culture dishes (107 cells/dish) coated successively with Poly-L-ornithine (MW ) 40 000, 15 µg/mL) and 10% fetal calf serum. The culture medium included a 1:1 mixture of DMEM and F-12 nutrient supplemented with glucose (33 mM), glutamine (2 mM), NaHCO3 (13 mM), HEPES buffer (5 mM, pH 7.4) penicillin-streptomycin (5 IU/mL-5 mg/mL) and a mixture of salt and hormones containing transferrin (100 µg/ mL), insulin (25 µg/mL), progesterone (20 nM), putrescine (60 nM) and Na2SeO3 (30 nM). Cultures were maintained for 7 days at 37 °C in a humidified atmosphere containing 5% CO2/95% air. At this stage, cultures were shown to contain at least 95% of neurons.16 2.4. Neuronal Secretome Recovery. Seven days after seeding, cortical neurons were washed 5 times in serum-free Eagle’s Basal Medium and incubated with a minimal volume of the same medium (6 mL per dish) for 12 h at 37 °C and 5% CO2 to allow extracellular accumulation of around 100 µg of proteins, in the absence or the presence of Brefeldin A (BFA, 1 µg/mL). Supernatant was then harvested, centrifuged at 300g for 10 min and then at 20 000g for 20 min to eliminate cells and cell debris, respectively. Neurons were washed with ice-cold PBS and scrapped off in 2 mL of lysis buffer containing Tris-HCl (50 mM, pH 7.4), EDTA (3 mM), MgCl2 (2 mM) and a cocktail of protease inhibitors (Roche Applied Science, Mannheim, Germany). Cells were homogenized 20 times on ice with a glassTeflon homogenizer and cytosol was cleared from nuclei and organelles by two successive centrifugations (300g for 10 min and 20 000g for 20 min, respectively). Protein concentration in neuron-conditioned media and neuronal cytosolic extracts was determined with the bicinchoninic acid method. 2.5. 1-D and 2-D Gel Electrophoresis. Native, immunodepleted, and retained fractions (400 µg proteins for SDS-PAGE and 100 µg proteins for 2-D electrophoresis) and neuronconditioned media or neuronal cytosolic extracts (100 µg of proteins) were concentrated by precipitation with 10% ice-cold trichloroacetic acid (2 h at 4 °C) and centrifuged at 38 000g for 25 min at 4 °C, and pellets were washed three times with diethyl ether. For SDS-PAGE, TCA precipitates were solubilized in SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 1% 2-mercaptoethanol, and 0.005% bromophenol blue) and proteins were resolved on 15% gels using the Protean II xi Cell system (Bio-Rad Laboratories, Hercules, CA). Gels were stained
Enhanced Detection of CNS Cell Secretome with the PageBlue Protein Staining Solution (Fermentas, Vilnius, Lithuania) and scanned using a computer-assisted densitometer (Epson Perfection V750 PRO). For 2-D electrophoresis, TCA precipitates were solubilized in isoelectrofocusing medium containing urea (7 M), thiourea (2 M), CHAPS (4%), ampholines (preblended, pI 3.5-9.5, 8 mg/mL, GE Healthcare, Uppsala, Sweden), dithiothreitol (DTT, 100 mM), tergitol NP7 (0.2%, Sigma) and traces of bromophenol blue.17 Proteins were first separated according to their isoelectric point along IPG strips (non linear gradient, pH 3-11, 18 cm long, GE Healthcare) using the IPGphor apparatus (GE Healthcare). Sample loading for the first dimension was performed by passive in-gel reswelling and proteins were focused under 5000 V until 70 000 V/h. After the first dimension, the IPG strips were equilibrated for 10 min in a buffer containing urea (6 M), Tris-HCl (50 mM, pH 6.8), glycerol (30%), SDS (2%), DTT (10 mg/mL) and bromophenol blue, and then for 15 min in the same buffer containing 15 mg/mL iodoacetamide instead of DTT. For the second dimension, strips were loaded onto vertical SDS polyacrylamide 11-17% gradient gels. The gels were stained with silver according to the procedure of Shevshenko et al.18 Gels were scanned using the computer-assisted densitometer, and analyzed using the SameSpots software (Non Linear Dynamics, Newcastle upon Tyne, U.K.) for gel morphing, background removal, spot detection and matching, and statistics. A minimum of four gels performed from different sets of cultured neurons were analyzed per experimental condition. 2.6. Protein Digestion. For proteins separated on 1-D gels, gel lanes were systematically cut into 68 regular pieces and destained with 3 washes with 50% acetonitrile. After protein reduction and alkylation (with 10 mM DTT for 15 min at 56 °C and 55 mM iodoacetamide for 30 min at room temperature, respectively), proteins were digested in-gel using trypsin (600 ng, Gold, Promega, Charbonnie`res, France), as previously described.18 Digest products were dehydrated in a vacuum centrifuge and reduced to 2 µL. For proteins separated on 2-D gels, spots of interest were excised and digested as previously described.17 2.7. LC-Q-TOF-MS/MS. Samples (1 µL) were analyzed online using nanoflow HPLC-nanoelectrospray ionization on a quadrupole time-of-flight (Q-TOF) mass spectrometer (QSTAR Pulsar-I, Applied Biosystems, Foster City, CA) coupled with an Ultimate 3000 HPLC (Dionex, Amsterdam, Netherland) using an uncoated silica PicoTip emitter with an outlet diameter of 8 µm (NewOjective, Woburn, MA). Desalting and preconcentration of samples were carried out online on a Pepmap precolumn (0.3 mm × 10 mm, Dionex). A gradient consisting of 0-40% B in 60 min, 40-80% B in 15 min (A ) 0.1% formic acid, 2% acetonitrile in water; B ) 0.1% formic acid in acetonitrile) at 300 nL/min was used to elute peptide from a Pepmap capillary (0.075 mm × 150 mm) reverse-phase column. Spectra were recorded using the Analyst QS 1.1 software (Applied Biosystems). Source parameters were adjusted as follows: ion spray voltage (IS), 1800 V; curtain gas (CUR), 25; declustering potential (DP), 75 V; focusing potential (FP), 265 V; declustering potential 2 (DP2), 15 V. Spectra were acquired with the instrument operating in the information-dependent acquisition mode throughout the HPLC gradient. Every 7 s, the instrument cycled through acquisition of a full-scan spectrum (1 s) and two MS/MS spectra (3 s each). Peptide fragmentation was performed using nitrogen gas on the most abundant doubly or triply charged ions detected in the initial MS scan,
research articles with a collision energy profile optimized according to peptide mass (manufacturer parameters) and an active exclusion time of 45 s. 2.8. Data Analysis and Protein Classification. All MS/MS spectra were searched against Homo sapiens entries of SwissProt/TrEMBL databases (http://www.expasy.ch, Uniprot 10.2, 44 495 647 entries), by using the Mascot v 2.1 algorithm (http:// www.matrixscience.com). All searches were carried out with peptide mass tolerance of (0.1 Da, fragment mass tolerance of (0.1 Da, carbamidomethyl as fixed modification of cysteines, methionine oxidation as variable modification, and one trypsin missed cleavage allowed. Peptides with scores greater than the identity score (p < 0.05) were considered as significant. All spectra were manually validated for proteins identified with less than three peptides. When the same set of peptides matched to multiple members of a protein family, Swiss-Prot entries were chosen upon TrEMBL entries. If several members matched within one database (Swiss-Prot or TrEMBL), the generic precursor was chosen upon isoforms or fragments, except if one or more significant peptides could discriminate them. MS/MS spectra were also searched against the H. sapiens entries of International Protein Index database (IPI, Version 3.17, ftp://ftp.ebi.ac.uk/pub/databases/IPI/old/HUMAN/) using the Mascot algorithm. This database is highly redundant and several IPI entries could be matched from one spectrum. Only peptides with p-values lower than 0.01 were automatically validated. IPI entries matched in this study were compared with the most complete human CSF map available11 and categorized according to their cellular distribution and function based on Gene Ontology (GO) analysis and annotations. Ontologies for cellular distribution were also searched for Swiss-Prot entries. 2.9. MALDI-TOF MS. Digest products of proteins separated onto 2-D gels were resuspended in 10 µL of formic acid (2%), desalted using Zip Tips C18 (Millipore, Molsheim, France), eluted with 10 µL of acetonitrile-trifluoroacetic acid (TFA), 50-0.1%, and concentrated to 2 µL. Aliquots (0.5 µL) were mixed with the same volume of R-cyano-4-hydroxy-transcinnamic acid (Sigma, 10 mg/mL in acetonitrile-TFA, 50-0.1%) and loaded on the target of an Ultraflex MALDI-TOF mass spectrometer (Bruker Daltonik, Bremen, Germany). Analyses were performed in reflectron mode with an accelerating voltage of 25 kV and a delayed extraction of 50 ns. Spectra were analyzed using the FlexAnalysis software (version 2.4, Bruker Daltonik) using the following parameters: resolution >5000, S/N > 2. Autoproteolysis products of trypsin (MW: 842.51, 1045.56, 2211.10, 2383.90) were used as internal calibrates and several contaminant peaks were removed before database interrogation: autoproteolysis products of trypsin (MW: 842.51, 870.54, 1045.56, 1791.72, 2211.10, 2383.90), keratin peptides (MW: 1066.44, 1232.62, 1277.70, 1475.78, 1993.97, 2383.95) and usual contaminants (MW: 982.47, 1107.55, 1109.56, 1157.51, 1165.58, 1179.59, 1193.61, 1235.60, 1265.63, 1302.69, 1307.67, 1365.63, 1434.84, 1493.79, 1707.76, 1716.84, 2225.10, 2299.95, 2718.09, 2705.15, 3223.25, 3312.31). Identification of proteins was performed using the Mascot software (version 2.1, Matrixscience) against H. sapiens or Mus musculus entries of the Swiss-Prot and TrEMBL databases. The following parameters were used for database interrogation: mass tolerance of 50 ppm; fixed modification, carbamidomethylation of cysteines; variable modification, oxidation of methionines; matching peptides with one missed cleavage accepted only when they included two consecutive basic residues or when arginine or lysine residues were followed by one or several acidic residues inside the Journal of Proteome Research • Vol. 7, No. 10, 2008 4411
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Figure 1. Increased sensitivity of CSF proteome analysis after immunodepletion of major plasma proteins. (A) Overview of the fractionation procedure used for CSF proteome analysis. Pooled CSF sample was concentrated to 4 mg/mL protein concentration by ultrafiltration through 3 kDa cutoff membrane and immunodepleted of 20 major plasma proteins using ProteoPrep 20 spin columns. Native, immunodepleted, and retained fractions (400 µg of proteins) were separated by SDS-PAGE. Each gel lane was systematically cut into 68 regular pieces and proteins were digested in-gel with trypsin. Trypsic peptides were analyzed by LC-MS/MS and proteins identified using the Mascot software against the Swiss-Prot/TrEMBL databases or the IPI database (version 3.17). (B) SDS-PAGE analysis of native CSF and CSF fractions obtained after immunodepletion. Proteins were stained with PageBlue. (C) Results of MS/MS analysis of the different CSF fractions. The diagram represents the number of nonredundant proteins identified in each fraction.
peptide aminoacid sequence. Mascot scores greater than 63 or 68 were considered as significant (p < 0.01) for Swiss-Prot or TrEMBL databases interrogation, respectively. When the same set of peptides matched to multiple members of a protein family, Swiss-Prot entries were chosen upon TrEMBL entries. If several members matched within one database (Swiss-Prot or TrEMBL), the generic precursor was chosen upon isoforms or fragments.
3. Results 3.1. Multiaffinity Depletion of 20 Abundant Plasma Proteins Enhances Protein Identification Rate in Human CSF Proteome Analysis. To remove 20 of the most abundant plasma proteins from human CSF, we have adapted for the first time the ProteoPrep 20 immunodepletion kit to CSF samples. This kit is the most complete plasma protein immunodepletion method available. Here, it was used on a CSF pool originating from 25 patients with various cognitive disorders to minimize interindividual variability and to get an unbiased view of the CSF proteome. Since protein concentration is 50-100 times lower in CSF than in plasma, the CSF pool was concentrated by ultrafiltration through a Centricon YM-3 Ultracell membrane with a nominal molecular weight cutoff of 3 kDa, yielding a 4 mg/mL protein concentration (Figure 4412
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1A). Concentrated fractions (800 µg of protein) were submitted to two depletion cycles on the ProteoPrep 20 column to ensure “complete” immunodepletion of plasma proteins. Separation of native CSF, immunodepleted, and retained fractions by SDS-PAGE revealed an important reduction of protein concentration dynamic range in the immunodepleted fraction, compared with native CSF (Figure 1B). Gel lanes were cut into 68 bands, digested with trypsin and analyzed by LCMS/MS. MASCOT interrogation for nonredundant proteins against Swiss-Prot/TrEMBL databases lead to the identification of 299 proteins in native CSF and 622 proteins in immunodepleted plus retained fractions. Of these, 302 were only detected in the immunodepleted fraction but not in native CSF (Figure 1C, see also Supplemental Table 1 in Supporting Information), underpinning the power of the immunodepletion approach used to increase sensitivity of CSF proteomic analyses. Moreover, the number of peptides matching for individual proteins was generally higher in the immunodepleted fraction, compared with native CSF (see Supplemental Table 1 in Supporting Information). Further supporting the efficacy of the ProteoPrep 20 method, the number of peptides matching the 20 plasma proteins retained by the column identified in the immunodepleted fraction by MS/MS was dramatically reduced and 11 of
Enhanced Detection of CNS Cell Secretome these proteins were not detectable in this fraction (see Supplemental Table 2 in Supporting Information). The number of CSF proteins identified was also markedly increased in samples depleted of 20 abundant plasma proteins (ProteoPrep 20) when compared to the corresponding samples depleted of 6 plasma proteins using the MARS 6 technology (see Supplemental Figure 1 in Supporting Information). An increased sensitivity of analyses was not only observed for proteins comigrating (same molecular weight) with proteins targeted by ProteoPrep 20 columns but not by MARS 6 columns (gel bands 1, 2 and 6, including TTR, alpha-1-acid-glycoprotein and alpha-2-macroglobulin, respectively) but also in gel areas lacking ProteoPrep 20-targeted proteins (bands 3, 4 and 5, see Supplemental Figure 1A and B in Supporting Information). ProteoPrep 20 also improved the confidence of identifications compared to MARS 6, as indicated by the larger number of peptides identified in each gel piece (see Supplemental Figure 1C in Supporting Information). 2-D gel analysis of native CSF and fractions obtained using ProteoPrep 20 confirmed efficient removal of the majority of major plasma proteins by this immunodepletion method, though TTR was still present in large amount in the immunodepleted fraction (Figure 2). Accordingly, immunodepletion resulted in a large increase in the amount of spots detectable on 2-D gels and identified by MALDI-TOF MS (depicted with arrowheads in Figure 2), indicating that this method can be relevant for differential proteomic analyses of CSF based on a 2-D MALDI approach. Moreover, six independent immunodepletions performed from the same CSF pool yielded a very similar 2-D protein profile in the depleted fractions, underscoring the high reproducibility of the ProteoPrep 20 approach (see Supplemental Figure 2 in Supporting Information). In the retained fraction, 234 proteins were identified. They comprise 117 proteins not found in the immunodepleted fraction (Figure 1C). These include almost exclusively proteins or fragments of proteins that are expected to bind to the column (see Materials and Methods). A majority of them were Ig isoforms (71 isoforms found). Only five proteins not targeted by the column (glutaminyl-peptide cyclotransferase, haptoglobin-related protein precursor, hemoglobin, plexin domaincontaining protein 2 and ribonuclease 4, see Supplemental Table 1C in Supporting Information) were detected in the retained fraction, but not in the immunodepleted fraction. Moreover, the 37 proteins identified only in the native sample (Figure 1C) also included a majority of Ig fragments. Although four of them (apoptosis-inducing factor 2, desmoplakin, isocitrate dehydrogenase, and pregnancy zone protein) were not targeted by the column, they were not identified in the immunodepleted fraction. 3.2. Toward an Enlarged CSF Proteome Map. During the past few years, several studies performed in different laboratories have provided more or less exhaustive proteomic maps of human CSF.9-11,18-25 A combination of several CSF studies based on MudPIT (multiple dimensional protein identification technology) systems (2-D-LC-ESI-IT, 2-D-LC-FTICR5,19 and 2-D-LC MALDI-TOF-TOF4) and search against the IPI database (version 3.01) generated a proteome data set including more than 2500 proteins.11 To compare our data with this data set, we analyzed them by automatic search against an updated version of the same database (version 3.17). We identified 1889 IPI entries from our MS/MS data: 693 of them (37% overlap) were referenced in this CSF proteome map (Supplemental Table 3 in Supporting Information).
research articles Comparing our data with previously published CSF proteome maps also revealed the presence of 22 novel proteins, which all were detected in the immunodepleted fraction (Table 1). Of these, 16 were identified with a single peptide (but with scores above the 99% confidence interval threshold -p < 0.01-) and only one was detected in native CSF, indicative of their low abundance in CSF. MS/MS spectra of these peptides, which were manually validated, are depicted on Supplemental Figure 3 in Supporting Information. Importantly, a majority of these newly identified proteins are known to exhibit extracellular localization or to be released in the extracellular medium following shedding of the ectodomain of a transmembrane precursor by secretase-like activities. They also include proteins already identified as secretory products of CNS cells (e.g., peroxiredoxin-6 and thrombospondin-1).13,14 In fact, GO analysis for cellular localization showed a large portion of proteins with predicted extracellular location (“extracellular region”, “extracellular region part”, “extracellular matrix” and “extracellular matrix part”) in our CSF proteome map (Figure 3A). Importantly, an even larger proportion of proteins exhibiting extracellular location (38% of the identified proteins) was found when Swiss-Prot/TrEMBL entries were taken into consideration (Figure 3B). Functional classification also revealed a majority (52%) of proteins involved in “binding” and a rather low amount of metabolic enzymes, pointing out limited contamination of our CSF samples by blood cells or by intracellular proteins following cell necrosis (Figure 3C). Moreover, a large portion of proteins identified in immunodepleted CSF are involved in biological processes important for CNS development and physiology such as “response to stimulus” and “biological adhesion” (Figure 3D). Finally, 107 CSF proteins identified in the immunodepleted fraction, versus only 44 in native CSF, are known to be expressed in the brain, as assessed by search in Swiss-Prot/TrEMBL annotations for tissue specificity, and might originate from CNS cells (see Figure 5A). 3.3. Identification of Proteins Released by Cultured Cortical Neurons. To further demonstrate the relevance of the fractionation procedure used herein to identify brain-derived proteins in CSF, especially those that are released by neurons and, thereby, could be potential biomarkers of CNS disorders, we sought to identify the major proteins secreted by cultured cortical neurons. Two-dimensional gel analysis of neuronconditioned media revealed the presence of numerous proteins, probably due to necrosis of a minority of neurons in these cultures (Figure 4A, B). For this reason, we did not systematically identify proteins present in neuron supernatants by LCMS/MS. Rather, to discriminate between proteins released by an active secretion mechanism and those recovered in neuronconditioned media as a consequence of cell necrosis, we first compared the 2-D protein pattern in conditioned-medium of neurons exposed or not to BFA (1 µg/mL, added during the secretion period) using the SameSpots software. BFA selectively blocks secretory vesicle assembly and, thus, the secretion of proteins through the vesicular pathway. Several spots or trains of spots detected in 2-D gels of extracellular medium of control neurons were absent or exhibited a reduced expression in the supernatant of BFA-treated neurons (Figure 4A, B). Only spots showing more than 30% decrease in their relative expression in the supernatant of BFA-treated neurons, compared with untreated neurons, were taken into consideration. The 34 unique proteins identified by peptide mass fingerprinting out of these spots are depicted in Table 2 and Supplemental Figure 4 in Supporting Information. They include several Journal of Proteome Research • Vol. 7, No. 10, 2008 4413
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Thouvenot et al. proteins known to be secreted by cells via the classical vesicular pathway and encompassing a signal peptide at their Nterminus (e.g., cystatin C (Cys C), apolipoprotein E (ApoE), matrix metalloprotease-inhibitor 2 (TIMP2), carboxypeptidase E (CBPE) and several complement subunits) as well as a larger set of proteins known be extracellularly released following proteolytic cleavage of the ectodomain of a membrane-bound or a transmembrane precursor. The majority of the proteins released by neurons upon proteolytic processing promote neuronal cell adhesion and migration (e.g., cadherin-2, -6 and -11, NRCAM and L1CAM), neurite outgrowth or axonal guidance (contactin-1 and -2, neurofascin and roundabout 2) and signaling (amyloid precursor protein (APP) and neogenin-1) (Table 2). Comparing the 2-D protein pattern of neuron-conditioned medium with that of neuronal cytosolic extract did not reveal extracellular enrichment of additional proteins, ruling out possible contribution of other (BFA-insensitive) secretion mechanisms to neuronal secretome. None of the proteins released by neurons either via the classical vesicular pathway or upon ectodomain shedding of a membrane precursor were detected in the cytosolic fraction, consistent with their intrinsic extracellular or membrane localization. 3.4. Enhanced Detection of CNS Cell Secretome in Immunodepleted CSF. Among the 34 proteins secreted by neurons identified, 27 were detected in human CSF following plasma protein immunodepletion, whereas only 21 of them were found in native CSF (Table 3). When proteins identified from two or more peptides were taken into consideration, 26 proteins (76%) secreted by neurons have been identified in fractionated CSF, versus 17 (50%) in native CSF. On the basis of the present and previous studies,13,14 a total of 70 nonredundant proteins were identified as secretory products of CNS cells, including not only neurons, but also astrocytes (the most abundant CNS cell population) and CECs (the main cell type contributing to CSF production). Of these, 52 (74%) were detected in immunodepleted CSF, whereas only 39 (56%) proteins were found in native CSF (Table 3 and Figure 5B). These values fall to 49 (70%) and 31 (44%), respectively, for proteins identified with 2 or more peptides.
4. Discussion
Figure 2. 2-D gel analysis of native CSF and CSF fractions obtained after immunodepletion of major plasma proteins. Native CSF, retained, and immunodepleted fractions (100 µg of proteins) were resolved on 2-D gels (pH 3-11, 11-17% gradient) and gels were stained with silver. Representative gels of 6 independent immunodepletion experiments are illustrated. Arrows indicate positions of proteins targeted by the ProteoPrep 20 column and arrowheads indicate the position of proteins not retained by the column and identified by peptide mass fingerprinting. Empty arrows indicate proteins that were identified in both immunodepleted CSF and neuron-conditioned media (see Figure 4). These include apolipoprotein E (APOE), beta-amyloid protein precursor (APP), amyloid-like protein 1 (APLP1), contactin 2 (CNTN2), cystatin C (CYSC), neural cell adhesion molecule 1 (NCA11) and L1 precursor (L1CAM), and SPARC-like protein 1 (SPRL1). 4414
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In the present study, we have used for the first time the most complete immunodepletion kit available, designed for 20 of the most abundant plasma proteins, to remove major plasma proteins from CSF samples prior to proteomic analysis. This immunodepletion method yielded an important reduction in the dynamic range of protein concentration in CSF and enabled us to identify a larger number of CSF proteins by MS/MS (622 proteins identified in fractionated CSF vs 299 proteins in native CSF). This approach also made possible the detection of proteins expressed at very low concentrations in CSF, including brain-derived proteins, and allowed enrichment of existing proteome maps of human CSF with previously unidentified proteins. 2-D gel analysis of CSF fractions obtained by immunodepletion demonstrated the high efficacy of the ProteoPrep 20 column to remove the majority of the most abundant CSF proteins, highlighting the relevance of this method, originally designed for plasma samples, to CSF proteome analysis. Among the 19 plasma proteins targeted by the column and identified in CSF (apolipoprotein B has never been found in our CSF samples), none of them except TTR were detectable on silver-
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Table 1. Newly Identified Proteins in Human CSF Swiss-Prot accession number
protein name
number of peptides
Mascot score
protein mass
cellular locali-zation
P31946* Q92485 Q8IUK8 P19835 P54108 Q969H8* Q08345 O00115 O75487 P62993* Q9BX67 O00187 Q8N0W4 P61970 P42785 P21810 P30041* Q5XPI4 O00241* Q9BUD6 P07996* O95497
14-3-3 protein beta/alpha Acid sphingomyelinase-like phosphodiesterase 3b precursor Cerebellin-2 precursor Bile salt-activated lipase precursor Cysteine-rich secretory protein 3 precursor Interleukin-25 Epithelial discoidin domain-containing receptor 1 precursor Deoxyribonuclease-2-alpha precursor Glypican-4 precursor Growth factor receptor-bound protein 2 Junctional adhesion molecule C precursor Mannan-binding lectin serine protease 2 precursor Neuroligin-4, X-linked precursor Nuclear transport factor 2 Lysosomal Pro-X carboxypeptidase precursor Biglycan precursor Peroxiredoxin-6 E3 ubiquitin-protein ligase subunit KPC1 Signal-regulatory protein beta-1 precursor Spondin-2 precursor Thrombospondin-1 precursor Pantetheinase precursor
5 1 1 1 1 2 1 1 1 1 1 2 1 1 3 1 1 1 2 1 1 1
205 76 69 63 57 62 59 42 88 50 104 107 57 60 139 60 73 50 126 46 89 39
28048 51237 24410 78638 28524 18897 102032 40069 63455 25304 35568 77224 92427 14640 56277 42027 25002 149960 43684 36335 133321 57716
C S M S S S M L M C M S M C L S C C M S S M
a Proteins identified for the first time in human CSF, compared with existing CSF proteomic maps, are depicted. Mascot scores and the number of matching peptides are indicated. Because of their very low concentration in CSF, many of them were only identified with one peptide. Note that all Mascot scores were greater than 33 (p < 0.01) and that all spectra have been manually validated. Cellular localization of proteins is also indicated (S, secreted; C, cytosolic; M, membrane; L, lysosome; proteins previously identified in post-mortem CSF are indicated by asteriks).
stained 2-D gels of the depleted fraction. Moreover, 11 of these proteins were not identified in the depleted fraction by MS/ MS, while the number of peptides matching the 8 other ones was markedly decreased, indicating an efficient (though incomplete) depletion. Plasma TTR is mainly produced and secreted by liver, whereas TTR contained in CSF originates from both plasma and brain where it is specifically synthesized and secreted by CECs.13 In fact, TTR was the most abundant protein identified in CEC secretome.13 Accordingly, TTR may be
overrepresented in CSF, compared with plasma, precluding its efficient removal from concentrated CSF by the ProteoPrep 20 column designed for plasma samples. This method was not designed for immunodepletion of the major brain-derived CSF protein, prostaglandin D synthase (PGDS, also known as β-trace protein). PGDS mainly originates from the brain where it is produced by choroid plexuses, leptomeningeal cells and oligodendrocytes.20 PGDS represents about 3% of the total CSF protein content21 and its concentra-
Figure 3. Gene Ontology annotations of CSF proteins. The pie charts depict the cellular components of the CSF proteins identified by search against IPI (v. 3.17) (A) or Swiss-Prot/TrEMBL (B) databases according to Gene Ontology (GO) analysis and annotations. Classification according to cellular location of Swiss-Prot/TrEMBL entries (B) shows a greater proportion (38%) of extracellular proteins, including extracellular matrix proteins. Classification according to molecular function (C) and biological process (D) of SwissProt/ TrEMBL entries is also depicted. Journal of Proteome Research • Vol. 7, No. 10, 2008 4415
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Figure 4. 2D-gel analysis of proteins secreted by primary cultured cortical neurons. Cortical neurons (10 millions neurons grown in 100-mm culture dishes) were exposed for 12 h in serum-free medium in the absence (Control) or presence of 1 µg/mL BFA. Cell-conditioned media and cytosolic extracts (100 µg proteins) were resolved on 2-D gels as described in the legend to Figure 2. Gels were stained with silver and analyzed using the SameSpot software. Representative 2-D gels of four independent experiments performed on different sets of cultured neurons are depicted. Proteins exhibiting more than 30% decrease in their relative expression in conditionedmedia from BFA-treated neurons, compared with control neurons or cytosolic extracts of control neurons, are indicated. Arrows indicate the position of proteins released by neurons via an active secretion mechanisms and identified in CSF. Arrowheads indicate the position of three proteins (cadherin-6 (CADH6), follistatin-related protein 5 (FSTL5), and roundabout 2 (ROBO2)) secreted by neurons but not identified in the present and previous CSF proteome analyses. 4416
Journal of Proteome Research • Vol. 7, No. 10, 2008
Thouvenot et al. tion is 35 times higher in CSF than in plasma.22 Accordingly, PGDS was found to be the most abundant protein in CSF following immunodepletion. Its high expression level in immunodepleted CSF samples may prevent detection and identification of less represented proteins comigrating with PGDS in conventional 2-D MALDI-based approaches. These limitations of the ProteoPrep 20 approach certainly outline the importance of developing methods specifically designed for immunodepletion of major CSF proteins, including PGDS and TTR, in order to further increase sensitivity of analyses toward weakly expressed proteins. Nonetheless, one should keep in mind that PGDS and TTR are themselves potential biomarkers of brain disorders. For instance, CSF TTR physically interacts with amyloid β (Aβ) peptides. This association prevents Aβ fibrillogenesis and inhibits Aβ deposition in the brain.23 Consistently, reduced TTR levels in CSF of patients with Alzheimer’s disease (AD) have been reported.24 A reduction in CSF concentration of TTR has also been associated with depression.25 In the same manner, PGDS was recently identified as a major Aβ chaperone that functions to prevent Aβ misfolding and aggregation in human CSF.26 Alteration of this function following, for instance, a decreased expression of PGDS, may be involved in the onset and progression of AD. Moreover, PGDS in CSF has also been proposed to be a useful marker for normal pressure hydrocephalus.27 Another limitation of the ProteoPrep 20 approach is “nonspecific” or indirect (via an intermediate protein partner) binding of CSF proteins to the column. Among the 234 proteins identified by MS/MS in the retained fraction, 97 proteins should theoretically not be retained by the column. Nonetheless, the Mascot score obtained for the majority of them was higher in the immunodepleted fraction than in the retained fraction or in native CSF, indicative of binding of a small portion of these proteins to the column. Moreover, none of the proteins solely detected either in the retained fraction or in native CSF were known to derive from the brain. Collectively, these findings indicate that investigating the immunodepleted fraction alone should not preclude identification of proteins originating from the brain and should be suitable for high-throughput CSF proteome profiling. The ProteoPrep 20 approach was more powerful than a previously employed immunodepletion technology (MARS 6) targeting only six major plasma proteins, highlighting the benefit of immunodepleting the largest number of abundant plasma proteins for deeper analysis of CSF proteome. Indeed, removal of a larger number of plasma proteins provided by the ProteoPrep 20 column, compared with other commercially available technologies, allowed analysis of more concentrated samples and increased the number of proteins identified by LC-MS/MS. Moreover, it will presumably increase the number of spots in gel areas otherwise occupied by these major proteins in 2-D MALDI approaches. Comparing our data with existing CSF proteome maps9-11,18-25 revealed the presence of 22 proteins in depleted CSF that were not detected in previous proteomic analyses performed on CSF samples originating from living patients. The majority of them are known to exhibit extracellular location or to correspond to membrane protein ectodomains. Interestingly, six of these proteins were previously identified in human postmortem CSF,28 suggesting that they might be released by damaged CNS cells. These include one of the 14-3-3 protein family isoforms (14-3-3 beta/alpha), which are considered as CSF biomarkers of Creutzfeldt-Jakob disease.29
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Enhanced Detection of CNS Cell Secretome a
Table 2. Proteins Identified in Cortical Neuron-Conditioned Medium protein identification
Secretogranin 1, SCG1
accession number
MW (Da)
pI
number of matching peptides
Neurotrophic Proteins 5.01 25
total number of peptides
Mascot score
sequence coverage
RMS error (ppm)
P16014
78036
38
262
42%
15
Amyloid beta A4 protein precursor, A4 Amyloid-like protein 1, APLP1 Cadherin 2, CADH2 Cadherin 6, CADH6 Cadherin 11, CAD11 Contactin 1, CNTN1 Contactin 2, CNTN2 Follistatin-related protein 5, FSTL5 Neogenin 1, NEO1 Neuronal cell adhesion molecule precursor, NRCAM Neural cell adhesion molecule 1, NCA11 Neural cell adhesion molecule L1 precursor, L1CAM Neural cell adhesion molecule L1-like protein precursor, CHL1 Neurofascin precursor, NFASC Roundabout 2, ROBO2 Semaphorin 7A, SEM7A
P12023
Cell Adhesion. Axon Guidance 87693 4.71 18
33
125
20%
11
Q03157
73390
5.46
16
30
150
24%
18
P15116 Q3KNY8 P55288 P12960 Q61330 Q8BFR2
100155 88355 88112 114172 113944 97066
4.60 4.82 4.72 5.80 7.55 5.51
11 9 6 14 11 13
16 22 7 36 15 23
116 76 69 103 107 122
10% 12% 10% 16% 12% 15%
15 16 9 11 15 12
Q7TQG5 Q810U4
160837 139290
6.22 5.61
17 14
25 29
153 100
16% 11%
11 18
P13595
120076
4.73
14
27
125
20%
16
Q6PGJ3
141798
5.64
12
21
108
13%
13
P70232
135985
5.46
26
38
224
25%
12
Q810U3
138688
5.84
10
13
102
10%
9
Q7TPD3 Q9QUR8
162002 74993
5.94 7.83
28 13
39 41
226 103
20% 21%
15 20
Calsyntenin 1, CSTN1 Calsyntenin 3, CSTN3 VPS10 domain-containing receptor SorCS1 precursor, SORC1 Receptor-type tyrosine-proteine phosphatase delta, PTPRD Receptor-type tyrosine-proteine phosphatase S, PTPRS Secreted protein acidic and rich in cysteine chain, SPRC
Q9EPL2 Q99JH7 Q9JLC4
Transduction and Modulation 4.82 23 30 5.17 15 26 7.36 8 10
226 149 87
21% 18% 7%
16 12 18
SPARC-like protein 1, SPRL1
Synaptic Signal 110086 107117 130690
Q5SPJ6
213498
6.13
13
14
138
10%
9
Q7TT17
168298
6.37
9
16
74
9%
11
P07214
35283
15
20
202
28%
14
P70633
73125
Matrix Proteins 4.51 16
22
187
28%
12
Carboxypeptidase E precursor, CBPE Complement C1qT4, C1QT4 Complement C4, CO4 Neuroserpin, NEUS
Q00493
53621
Q8R066 P01029 O35684
35207 192885 46375
Cystatin C, CYSC Metalloproteinase inhibitor 2 chain 1, TIMP2
P21460 P25785
Apoliporotein E, APOE Cp protein, CERU Transferrin, TRFE Insulin-like growth factor binding protein 2, IBP2
P08226 Q6P5C8 Q921I1 P47877
4,77
Proteases 5.07
19
28
219
35%
12
9.10 7.38 4.66
14 7 15
24 9 22
182 71 175
40% 7% 35%
10 15 16
33 24
164 126
66% 30%
17 20
31 28 24 18
214 146 218 123
47% 16% 29% 38%
14 15 20 10
Proteases and Protease Inhibitors 15749 9.18 14 24996 7.45 10
35901 121872 78841 33909
Carrier Proteins 5.56 19 5.53 16 6.94 17 7.81 9
a Proteins that were detected in 2-D gels of supernatant of control neurons and that were absent or that exhibited a decreased expression in the supernatant of BFA-treated neurons (34 spots or trains of spots) were identified by peptide mass fingerprinting. When several spots were identified as a single protein, the sequence coverage and MASCOT score resulting from the analysis of the major spot is indicated. RMS error: root mean squared error of mass deviations.
Intriguingly, comparing our data with the two most complete CSF proteome data sets generated to date using multidimen-
sional separation technologies and MS/MS11,30 by search against the IPI human protein database indicated large differJournal of Proteome Research • Vol. 7, No. 10, 2008 4417
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Thouvenot et al. a
Table 3. CNS Cell-Derived Proteins Identified in Human CSF Proteins protein identification
CNS Cell Secretome mouse accession number
human accession number
Netrin 1, NET1 Pigment epithelium-derived factor, PEDF Secretogranin 1 WNT-7B WNT-10A
O09118 P97298
P36955
P16014 P28047 P70701
P05060 -
Amyloid beta A4 protein precursor, APP Amyloid-like protein 1, APLP1 Cadherin 2, CADH2 Cadherin 6, CADH6 Cadherin 11, CAD11 Contactin 1, CNTN1 Contactin 2, CNTN2 Follistatin-related protein 5, FSTL5 Neogenin 1, NEO1 Neuronal cell adhesion molecule precursor, NRCAM Neural cell adhesion molecule 1, NCA11 Neural cell adhesion molecule L1 precursor, L1CAM Neural cell adhesion molecule L1-like protein precursor, CHL1 Neurofascin precursor, NFASC Roundabout 2, ROBO2 Semaphorin 7A, SEM7A
P12023
Human CSF Number of Matching Peptides
choroid plexus epithelial cells
astrocytes
Neurotrophic Proteins + + + +
-
neurons
immunodepleted
native
immunodepleted
native
-
+
+
20
18
+ -
+ -
+ -
3 -
1 -
+
21
1
Cell Adhesion Molecules and Axonal Guidance Proteins P05067 + +
Q03157 P15116 Q3KNY8 P55288 P12960 Q61330 Q8BFR2
P51693 P19022 P55287 Q12860 Q02246 -
-
-
+ + + + + + +
+ + + + + -
+ + + -
15 6 4 31 27 -
6 8 5 -
Q7TQG5 Q810U4
Q92859 Q92823
-
-
+ +
+ +
+
15 28
4
P13595
P13591
-
-
+
+
+
19
3
Q6PGJ3
P32004
-
-
+
+
-
3
-
P70232
O00533
-
-
+
+
+
30
16
Q810U3 Q7TPD3 Q9QUR8
O94856 O75326
-
-
+ + +
+ +
+
7 16
1
Calsyntenin 1, CSTN1 Calsyntenin 3, CSTN3 VPS10 domain-containing receptor SorCS1 precursor, SORC1 Receptor-type tyrosine-proteine phosphatase delta, PTPRD Receptor-type tyrosine-proteine phosphatase S, PTPRS Secreted protein acidic and rich in cysteine chain, SPRC
Q9EPL2 Q99JH7 Q9JLC4
Synaptic Signal Transduction and Modulation O94985 + + +
+ -
+ -
28 -
7 -
Q5SPJ6
P23468
-
-
+
+
-
1
-
Q7TT17
Q13332
-
-
+
+
-
3
-
P07214
P09486
+
+
+
+
+
7
2
Collagen alpha 1 (I), CO1A1 Collagen alpha 1 (I), fragment (C-terminus), CO1A1 Collagen alpha 1 (I), fragment, CO1A1 Collagen alpha 1 (III), CO3A1 Collagen alpha 1 (III), fragment (C-terminus), CO3A1 Collagen alpha 1 (IV), CO4A1 Collagen alpha 2 (I) precursor, CO1A2 Collagen alpha 2 (I) fragment (C-terminus), CO1A2 Fibronectin 1 chain 1, FINC Fibulin 1, FBLN1 Perlecan, PGBM Procollagen alpha 2 (V), CO5A2 Procollagen C-proteinase enhancer protein, PCOC1 SPARC-like protein 1, SPRL1 Thrombospondin 1, TSP-1
P11087 Q60785
Matrix and Cell-Matrix Interacting Proteins P02452 + P02452 + -
+ +
+ +
2 2
1 1
Q99LL6
P02452
+
-
-
+
+
2
1
P08121 Q8BJU6
P02461 P02461
+ +
-
-
+ +
-
4 4
-
P02463 Q01149
P08123
+ +
-
-
+
-
3
-
Q91VL4
P08123
+
-
-
+
-
3
-
P11276 Q08879 Q05793 Q61431
P02751 P23142 P98160 -
+ + + +
-
-
+ + + -
+ + + -
24 13 11 -
11 4 7 -
Q61398
Q15113
+
-
-
+
+
6
7
P70633 P35441
Q14515 P07996
+
-
+ -
+ +
+ -
23 1
2 -
Q00493
P16870
+
Proteases +
+
+
+
11
2
Q8C243 Q8R066 P01027 P01029 P33434
P07339 Q9BXJ3 P01024 P0C0L5 P08253
+ + +
+ -
+ + -
+ + + + +
+ + + +
19 1 16 7 17
14 25 26 2
Carboxypeptidase E precursor, CBPE Cathepsin D precursor, CATD Complement C1qT4, C1QT4 Complement C3, CO3 Complement C4, CO4 Matrix metalloproteinase 2, MMP2
4418
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Enhanced Detection of CNS Cell Secretome Table 3. Continued Proteins protein identification
Matrix metalloproteinase 3, MMP3 Neuroserpin, NEUS Subtilisin proprotein convertase, PCSK5 Complement factor H, CFH Cystatin C, CYSC Metalloproteinase inhibitor 1 chain 1, TIMP1 Metalloproteinase inhibitor 2 chain 1, TIMP2 Plasminogen activator inhibitor1, PAI1 Serine protease inhibitor 22, SPI22 Serine protease inhibitor 6, SPI6 Apoliporotein E, APOE Ceruloplasmin, CERU Insulin-like growth factor binding protein 2, IBP2 Insulin-like growth factor binding protein 7, IBP7 Neutrophil gelatinase-associated protein, NGAL Transcobalamin II, TCO2 Transferrin, TRFE Transthyretin precursor, TTR Beta-2 microglobulin, B2MG Cyclophilin-associated protein, CAP Chitinase 3-like protein-1 chain 1, CH3L1 Pentraxin 3, PTX3
CNS Cell Secretome mouse accession number
human accession number
Human CSF Number of Matching Peptides
choroid plexus epithelial cells
astrocytes
neurons
immunodepleted
native
immunodepleted
native
P28862
-
+
-
-
-
-
-
-
O35684 Q04592
Q99574 -
+
-
+ -
+ -
+ -
4 -
1 -
P06909 P21460 P12032
P08603 P01034 P01033
Protease Inhibitors + + + + +
+ -
+ + +
+ + +
57 7 7
13 10 5
P25785
P16035
+
+
+
+
+
9
3
P22777
-
+
-
-
-
-
-
-
Q91WP6
-
+
-
-
-
-
-
-
O08797
-
+
-
-
-
-
-
-
P08226 Q6P5C8 P47877
P02649 P00450 P18065
Carrier Proteins + + + + + +
+ + +
+ +
+ + +
26 10
26 3 3
Q61581
Q16270
+
-
-
+
+
14
10
P11672
P80188
+
+
-
+
-
1
-
O88968 Q921I1 P07309
P20062 P02787 P02766
+ + +
+ -
+ -
+ + +
+ + +
3 16 9
1 51 13
P01887 O35649
P61769 -
+ -
+ -
5 -
5 -
Q61362
P36222
+
+
-
+
+
19
17
P48759
-
+
+
-
-
-
-
-
Inflammation Related Proteins + + + -
a Proteins identified as secretion products of cultured neurons (present study), astrocytes or CECs (previous studies) are listed. Their identification and the number of matching peptides in native and immunodepleted CSF is also indicated.
ences in CSF proteins identified. Among the 1889 IPI entries identified herein, only 693 (37%) can be found in the list generated by Pan et al., which comprises 2594 IPI entries.11 Moreover, a 49% overlap between our CSF proteome and the most recently published CSF data set of Zougman et al. was found after removing from our protein list proteins identified with one peptide, keratins and redundancy.30 The partial overlap between different CSF proteome maps may be due to differences in sample collection and processing, fractionation strategy, mass spectrometry technologies, algorithms (Mascot vs SEQUEST) and criteria for database interrogation and, finally, the different versions of IPI database used (3.17 vs 3.01 in the data set created by Pan et al.). Of note, this discrepancy cannot be explained by blood contamination during lumbar punctures, as shown by the lack of detection of APOB in our CSF samples. The partial overlap between different CSF proteome maps also points out requirement of standard procedure based on a unique database for establishment of a reference CSF proteome map. In this regard, search against the IPI database generated important redundancy compared with Swiss-Prot/TrEMBL database interrogation: with the same set of MS/MS data, we matched 1889 IPI entries by search against the IPI database, whereas 659 unique proteins were identified in fractionated and native CSF by search against Swiss-Prot/TrEMBL databases. Redundancy extraction from recent IPI database using bioinformatics tools or search against well annotated and low-
redundancy databases such as Swiss-Prot/TrEMBL would thus be probably more relevant to combine multicentric data sets to generate an accurate and standard CSF proteome map. CSF is a key sample in the research of biomarkers for brain disorders and numerous attempts using various proteomic platforms based on different separation strategies and mass spectrometry technologies have been made to achieve this goal. A major challenge in these studies is to identify brain-derived proteins that are typically low in abundance and whose expression is modified in pathological situations. We investigated for the first time the neuronal secretome and identified 34 proteins secreted via the classical vesicular pathway or released by cells following shedding of ectodomain of membrane precursors by proteases. Only 17 of them were previously identified in a global analysis of the cortical neuron proteome that detected more than 4500 unique proteins,31 pointing out the specificity of secretome versus global cell proteome and its relevance for biomarker discovery.32,33 The proteins secreted by cortical neurons identified in our study are known to play critical roles in neurite outgrowth and axonal guidance, modulation of synaptic transmission, cell adhesion or protein degradation and some of them have been implicated in neurodegenerative diseases such as AD (e.g., apolipoprotein E and APP). Immunodepletion of CSF with the ProteoPrep 20 method not only enhanced the identification of major secretory prodJournal of Proteome Research • Vol. 7, No. 10, 2008 4419
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Thouvenot et al. et de la Technologie (ACI Neurosciences 2004), INCA and La Re´gion Languedoc-Roussillon. Mass spectrometry analyses were performed using the facilities of the Proteomic Platform of Montpellier Languedoc-Roussillon. The authors also thank Dr. Sylvain Lehmann for his assistance in the MARS 6 technology. E.T. was supported by fellowships from the Fondation pour la Recherche Me´dicale and INSERM.
Figure 5. Enhanced detection of CNS cell secretome in immunodepleted CSF. Tissue specificity annotations of proteins identified by search against the Swiss-Prot/TrEMBL database (http:// www.expasy.org/uniprot/) show a greater proportion of proteins known to be expressed in the CNS in the immunodepleted fraction (107 proteins, 21%) compared to native CSF (44 proteins, 15%) (A). Search for proteins secreted by CNS cells in native and immunodepleted CSF also reveals enhanced detection of CNS cell secretome in immunodepleted CSF (B).
ucts of neurons but also provided a better coverage of the previously described astrocytic and CP cell secretomes in CSF proteomic analysis.13,14 Assuming that these cell populations exhibit the same protein secretion profiles in adult human CNS in situ, these findings show that the ProteoPrep 20 depletion method enables better characterization of CNS cell-derived proteins in CSF proteome analysis. Consistently, Swiss-Prot/ TrEMBL annotations for tissue specificity suggested that 107 CSF proteins identified in the immunodepleted fraction were known to be produced in the CNS, compared to only 44 in native CSF. However, we cannot rule out that a significant share of some of these proteins also originates from plasma.
Conclusion In summary, we have demonstrated that a method initially designed for immunodepletion of 20 abundant plasma proteins markedly increases sensitivity of CSF proteome analysis and improves the detection of proteins actually released by CNS cells, including neurons, glial cells and choroid plexus cells, in CSF. This approach allowed the identification of several proteins associated with inflammatory conditions in CSF, which thereby are biomarker candidates of CNS diseases associated with inflammatory processes. These include, for instance, NGAL, a protein secreted in very low amounts by astrocytes under control conditions and whose synthesis and secretion are greatly increased upon proinflammatory treatments.14 This highlights how an efficient depletion method targeting a “large” number of abundant plasma proteins is a necessary step in sensitive CSF proteome profiling for specific biomarker discovery. Abbreviations: AD, Alzheimer’s disease; Aβ, amyloid β; APP, amyloid precursor protein; BFA, brefeldin A; CEC, choroidal epithelial cell; CP, choroid plexus; CSF, cerebrospinal fluid; GO, Gene Ontology; NGAL, neutrophil gelatinase-associated lipocalin; PGDS, prostaglandin D synthase; TTR, transthyretin.
Acknowledgment. This work was supported by grants from CNRS, INSERM, the French Ministe`re de la Recherche 4420
Journal of Proteome Research • Vol. 7, No. 10, 2008
Supporting Information Available: Three supplemental tables and four supplemental figures that support the data presented are provided. These include: Supplemental Table 1, total list of unique proteins and peptides identified by LC-MS/ MS in this study, peptide sequences, masses and charges, and Mascot scores; Supplemental Table 2, number of peptides deriving from the 20 proteins targeted by the ProteoPrep 20 column identified in native and immunodepleted CSF fractions; Supplemental Table 3, total list of IPI entries identified in CSF by LC-MS/MS; Supplemental Figure 1, comparison of ProteoPrep 20 and MARS 6 immunodepletion techniques; Supplemental Figure 2, 2-D gels of the immunodepleted fractions obtained in six independent immunodepletion experiments performed from the same CSF pool; Supplemental Figure 3, list of annotated MS/MS spectra obtained for CSF proteins identified from a single peptide; Supplemental Figure 4, list of annotated MALDI-TOF MS spectra obtained for proteins identified in cortical neuron-conditioned medium. This material is available free of charge via the Internet at http:// pubs.acs.org. References (1) Zhang, J.; Goodlett, D. R.; Montine, T. J. Proteomic biomarker discovery in cerebrospinal fluid for neurodegenerative diseases. J. Alzheimer’s Dis. 2005, 8 (4), 377–86. (2) Choudhary, J.; Grant, S. G. Proteomics in postgenomic neuroscience: the end of the beginning. Nat. Neurosci. 2004, 7 (5), 440– 5. (3) Hu, S.; Loo, J. A.; Wong, D. T. Human body fluid proteome analysis. Proteomics 2006, 6 (23), 6326–53. (4) Abdi, F.; Quinn, J. F.; Jankovic, J.; McIntosh, M.; Leverenz, J. B.; Peskind, E.; Nixon, R.; Nutt, J.; Chung, K.; Zabetian, C.; Samii, A.; Lin, M.; Hattan, S.; Pan, C.; Wang, Y.; Jin, J.; Zhu, D.; Li, G. J.; Liu, Y.; Waichunas, D.; Montine, T. J.; Zhang, J. Detection of biomarkers with a multiplex quantitative proteomic platform in cerebrospinal fluid of patients with neurodegenerative disorders. J. Alzheimer’s Dis. 2006, 9 (3), 293–348. (5) Xu, J.; Chen, J.; Peskind, E. R.; Jin, J.; Eng, J.; Pan, C.; Montine, T. J.; Goodlett, D. R.; Zhang, J. Characterization of proteome of human cerebrospinal fluid. Int. Rev. Neurobiol. 2006, 73, 29–98. (6) Sihlbom, C.; Davidsson, P.; Nilsson, C. L. Prefractionation of cerebrospinal fluid to enhance glycoprotein concentration prior to structural determination with FT-ICR mass spectrometry. J. Proteome Res. 2005, 4 (6), 2294–301. (7) Ramstrom, M.; Hagman, C.; Mitchell, J. K.; Derrick, P. J.; Hakansson, P.; Bergquist, J. Depletion of high-abundant proteins in body fluids prior to liquid chromatography fourier transform ion cyclotron resonance mass spectrometry. J. Proteome Res. 2005, 4 (2), 410–6. (8) Hu, Y.; Malone, J. P.; Fagan, A. M.; Townsend, R. R.; Holtzman, D. M. Comparative proteomic analysis of intra- and interindividual variation in human cerebrospinal fluid. Mol. Cell. Proteomics 2005, 4 (12), 2000–9. (9) Ogata, Y.; Charlesworth, M. C.; Muddiman, D. C. Evaluation of protein depletion methods for the analysis of total-, phospho- and glycoproteins in lumbar cerebrospinal fluid. J. Proteome Res. 2005, 4 (3), 837–45. (10) Shores, K. S.; Knapp, D. R. Assessment approach for evaluating high abundance protein depletion methods for cerebrospinal fluid (CSF) proteomic analysis. J. Proteome Res. 2007, 6 (9), 3739–51. (11) Pan, S.; Zhu, D.; Quinn, J. F.; Peskind, E. R.; Montine, T. J.; Lin, B.; Goodlett, D. R.; Taylor, G.; Eng, J.; Zhang, J. A combined dataset of human cerebrospinal fluid proteins identified by multidimensional chromatography and tandem mass spectrometry. Proteomics 2007, 7 (3), 469–73.
research articles
Enhanced Detection of CNS Cell Secretome (12) Shores, K. S.; Udugamasooriya, D. G.; Kodadek, T.; Knapp, D. R. Use of Peptide analogue diversity library beads for increased depth of proteomic analysis: application to cerebrospinal fluid. J. Proteome Res. 2008, 7 (5), 1922–31. (13) Thouvenot, E.; Lafon-Cazal, M.; Demettre, E.; Jouin, P.; Bockaert, J.; Marin, P. The proteomic analysis of mouse choroid plexus secretome reveals a high protein secretion capacity of choroidal epithelial cells. Proteomics 2006, 6 (22), 5941–52. (14) Lafon-Cazal, M.; Adjali, O.; Galeotti, N.; Poncet, J.; Jouin, P.; Homburger, V.; Bockaert, J.; Marin, P. Proteomic analysis of astrocytic secretion in the mouse. Comparison with the cerebrospinal fluid proteome. J. Biol. Chem. 2003, 278 (27), 24438–48. (15) Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Measurement of protein using bicinchoninic acid. Anal. Biochem. 1985, 150 (1), 76–85. (16) Weiss, S.; Sebben, M.; Kemp, D. E.; Bockaert, J. Serotonin 5-HT1 receptors mediate inhibition of cyclic AMP production in neurons. Eur. J. Pharmacol. 1986, 120 (2), 227–30. (17) Delcourt, N.; Thouvenot, E.; Chanrion, B.; Galeotti, N.; Jouin, P.; Bockaert, J.; Marin, P. PACAP type I receptor transactivation is essential for IGF-1 receptor signalling and antiapoptotic activity in neurons. EMBO J. 2007, 26 (6), 1542–51. (18) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 1996, 68 (5), 850–8. (19) Pan, S.; Wang, Y.; Quinn, J. F.; Peskind, E. R.; Waichunas, D.; Wimberger, J. T.; Jin, J.; Li, J. G.; Zhu, D.; Pan, C.; Zhang, J. Identification of glycoproteins in human cerebrospinal fluid with a complementary proteomic approach. J. Proteome Res. 2006, 5 (10), 2769–79. (20) Urade, Y.; Kitahama, K.; Ohishi, H.; Kaneko, T.; Mizuno, N.; Hayaishi, O. Dominant expression of mRNA for prostaglandin D synthase in leptomeninges, choroid plexus, and oligodendrocytes of the adult rat brain. Proc. Natl. Acad. Sci. U.S.A. 1993, 90 (19), 9070–4. (21) McArthur, J.; Hill, J.; Paech, M. J.; Dodd, P. H.; Bennett, E. J.; Holden, J. Cerebrospinal fluid and serum concentrations of betatrace protein during pregnancy. Anaesthesia 2005, 60 (2), 163–7. (22) Melegos, D. N.; Freedman, M. S.; Diamandis, E. P. Prostaglandin D synthase concentration in cerebrospinal fluid and serum of patients with neurological disorders. Prostaglandins 1997, 54 (1), 463–74.
(23) Giunta, S.; Valli, M. B.; Galeazzi, R.; Fattoretti, P.; Corder, E. H.; Galeazzi, L. Transthyretin inhibition of amyloid beta aggregation and toxicity. Clin. Biochem. 2005, 38 (12), 1112–9. (24) Sousa, J. C.; Cardoso, I.; Marques, F.; Saraiva, M. J.; Palha, J. A. Transthyretin and Alzheimer’s disease: where in the brain. Neurobiol. Aging 2007, 28 (5), 713–8. (25) Sullivan, G. M.; Mann, J. J.; Oquendo, M. A.; Lo, E. S.; Cooper, T. B.; Gorman, J. M. Low cerebrospinal fluid transthyretin levels in depression: correlations with suicidal ideation and low serotonin function. Biol. Psychiatry 2006, 60 (5), 500–6. (26) Kanekiyo, T.; Ban, T.; Aritake, K.; Huang, Z. L.; Qu, W. M.; Okazaki, I.; Mohri, I.; Murayama, S.; Ozono, K.; Taniike, M.; Goto, Y.; Urade, Y. Lipocalin-type prostaglandin D synthase/beta-trace is a major amyloid beta-chaperone in human cerebrospinal fluid. Proc. Natl. Acad. Sci. U.S.A. 2007, 104 (15), 6412–7. (27) Mase, M.; Yamada, K.; Shimazu, N.; Seiki, K.; Oda, H.; Nakau, H.; Inui, T.; Li, W.; Eguchi, N.; Urade, Y. Lipocalin-type prostaglandin D synthase (beta-trace) in cerebrospinal fluid: a useful marker for the diagnosis of normal pressure hydrocephalus. Neurosci. Res. 2003, 47 (4), 455–9. (28) Burgess, J. A.; Lescuyer, P.; Hainard, A.; Burkhard, P. R.; Turck, N.; Michel, P.; Rossier, J. S.; Reymond, F.; Hochstrasser, D. F.; Sanchez, J. C. Identification of brain cell death associated proteins in human post-mortem cerebrospinal fluid. J. Proteome Res. 2006, 5 (7), 1674–81. (29) Shiga, Y.; Wakabayashi, H.; Miyazawa, K.; Kido, H.; Itoyama, Y. 14-3-3 protein levels and isoform patterns in the cerebrospinal fluid of Creutzfeldt-Jakob disease patients in the progressive and terminal stages. J. Clin. Neurosci. 2006, 13 (6), 661–5. (30) Zougman, A.; Pilch, B.; Podtelejnikov, A.; Kiehntopf, M.; Schnabel, C.; Kumar, C.; Mann, M. Integrated analysis of the cerebrospinal fluid peptidome and proteome. J. Proteome Res. 2008, 7 (1), 386– 99. (31) Yu, L. R.; Conrads, T. P.; Uo, T.; Kinoshita, Y.; Morrison, R. S.; Lucas, D. A.; Chan, K. C.; Blonder, J.; Issaq, H. J.; Veenstra, T. D. Global analysis of the cortical neuron proteome. Mol. Cell. Proteomics 2004, 3 (9), 896–907. (32) Lescuyer, P.; Hochstrasser, D.; Rabilloud, T. How shall we use the proteomics toolbox for biomarker discovery. J. Proteome Res. 2007, 6 (9), 3371–6. (33) Andrade, E. C.; Krueger, D. D.; Nairn, A. C. Recent advances in neuroproteomics. Curr. Opin. Mol. Ther. 2007, 9 (3), 270–81.
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Journal of Proteome Research • Vol. 7, No. 10, 2008 4421
