Identification of Brain Cell Death Associated Proteins in Human Post

Proteomic Analysis of Brain and Cerebrospinal Fluid from the Three Major Forms of Neuronal Ceroid Lipofuscinosis Reveals Potential Biomarkers. David E...
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Identification of Brain Cell Death Associated Proteins in Human Post-mortem Cerebrospinal Fluid Jennifer A. Burgess,†,# Pierre Lescuyer,†,# Alexandre Hainard,† Pierre R. Burkhard,‡ Natacha Turck,† Philippe Michel,§ Joe1 l S. Rossier,§ Fre´ de´ ric Reymond,§ Denis F. Hochstrasser,†,|,⊥ and Jean-Charles Sanchez*,† Biomedical Proteomics Research Group, Department of Structural Biology and Bioinformatics, Faculty of Medicine, University of Geneva, Geneva, Switzerland, Department of Neurology, Geneva University Hospitals and Medical School, Geneva, Switzerland, DiagnoSwiss SA, Monthey, Switzerland, Clinical Chemistry Laboratory, Geneva University Hospital, Geneva, Switzerland, and Pharmacy Section, Faculty of Sciences, University of Geneva, Geneva, Switzerland Received April 10, 2006

Following any form of brain insult, proteins are released from damaged tissues into the cerebrospinal fluid (CSF). This body fluid is therefore an ideal sample to use in the search for biomarkers of neurodegenerative disorders and brain damage. In this study, we used human post-mortem CSF as a model of massive brain injury and cell death for the identification of such protein markers. Pooled post-mortem CSF samples were analyzed using a protocol that combined immunoaffinity depletion of abundant CSF proteins, off-gel electrophoresis, SDS-PAGE and protein identification by LC-MS/MS. A total of 299 proteins were identified, of which 172 proteins were not previously described to be present in CSF. Of these 172 proteins, more than 75% have been described as intracellular proteins suggesting that they were released from damaged cells. Immunoblots of a number of proteins were performed on individual post-mortem CSF samples and confirmed elevated concentrations in post-mortem CSF compared to ante-mortem CSF. Interestingly, among the proteins specifically identified in the postmortem CSF, several have been previously described as biochemical markers of brain damage. Keywords: brain damage • neurodegeneration • neurodegenerative disease • cerebrospinal fluid • biomarker

Introduction Body fluids are a valuable source of diagnostic and prognostic disease markers. One reason for this is that they may contain leakage products from various tissues, including proteins normally located within cells that are released into body fluids following cell damage or death.1 Body fluids may also contain proteins that are secreted in response to pathological processes. Such proteins include those that are abnormally expressed and secreted by the affected tissue, inflammatory markers secreted by the liver, immunoglobulins or cytokines secreted by the immune system, vasoactive compounds or mediators of axonal growth involved in regeneration. The detection and measurement in body fluids of these tissue leakage products and secreted proteins can therefore be useful to help diagnose a pathological process and to monitor its * To whom correspondence should be addressed. E-mail: [email protected]. † Biomedical Proteomics Research Group, Department of Structural Biology and Bioinformatics, Faculty of Medicine, University of Geneva. ‡ Department of Neurology, Geneva University Hospitals and Medical School. § DiagnoSwiss SA. | Clinical Chemistry Laboratory, Geneva University Hospital. ⊥ Pharmacy Section, Faculty of Sciences, University of Geneva. # These authors contributed equally to this work.

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evolution. In this context, cerebrospinal fluid (CSF) represents an ideal sample to use in the search for biomarkers associated with brain damage. Owing to the close proximity of CSF to the brain, changes that occur in the protein composition of CSF often reflect changes that occur or have occurred in the brain.2,3 In recent years, advances in proteomics and related technologies have enabled the identification of human CSF proteins in ever decreasing concentration. Proteomic analyses of human CSF have included 2-DE,4-10 liquid-phase IEF together with 2-DE11,12 and 2D-LC of the peptides resulting from enzymatic cleavage of unseparated CSF proteins.13-16 Using the latter approach, Zhang and colleagues identified more than 300 proteins in human CSF.15 A number of studies, which aimed at identifying potential biomarkers of various neurological disorders by comparative analyses of CSF samples from healthy and diseased subjects, have also been published. A recent publication reported the relative quantification of 163 proteins in CSF samples from well-characterized Alzheimer’s disease patients and age-matched controls using the ICAT technique.16 In a previous publication, we presented the use of ventricular post-mortem CSF as a model of massive brain insult.17 It was hypothesized that protein leakage mechanisms associated with the global brain necrosis that follows death may share similarities with those occurring in brain lesions related to neurological 10.1021/pr060160v CCC: $33.50

 2006 American Chemical Society

Brain Cell Proteins in Post-mortem CSF

disorders. According to this hypothesis, post-mortem CSF could be a useful source of potential protein markers of brain damage. Ante-mortem and post-mortem CSF samples were compared using 2-DE gels and 12 proteins showing increased levels in post-mortem CSF were identified. From these proteins, three have been further validated as potential biomarkers for the early diagnosis of stroke: heart fatty acid-binding protein (H-FABP), nucleoside diphosphate kinase A (NDKA) and DJ-1/PARK7.18-20 H-FABP was also shown to be a potential diagnostic marker of Creutzfeldt-Jakob disease (CJD) and other neurodegenerative dementias.21,22 In addition, changes in the pattern of posttranslational modifications of prostaglandin D2 synthase were observed in post-mortem CSF compared to ante-mortem CSF. Further analysis indicated that similar changes were associated with neurological disorders and, in particular, neurodegenerative dementias.23 Taken together these data strongly support human post-mortem CSF as a useful sample source for the identification of proteins associated with brain damage and cell death. In the current work, we have used an alternative method to 2-DE in order to further characterize the human post-mortem CSF proteome. A pool of post-mortem CSF samples (n ) 5) was analyzed using a four step protocol: (i) immunodepletion of abundant CSF proteins (albumin, IgG, IgA, transferrin, antitrypsin, and haptoglobin), (ii) fractionation of CSF proteins according to their pI using off-gel electrophoresis (OGE),24 (iii) analysis of fractions from OGE by SDS-PAGE, (iv) protein identification by LC-MS/MS. A total of 299 proteins were identified, of which 172 proteins were not previously described to be present in CSF. Selected proteins that were identified in post-mortem CSF were validated using Western blots and ELISA of individual post-mortem and ante-mortem CSF samples. The potential interest of proteins identified in post-mortem CSF as biomarkers of brain damage will be discussed.

Experimental Procedures Materials. All chemicals, unless otherwise stated, were purchased from Sigma Aldrich (St. Louis, MI) and were of the highest purity available. CH3CN was purchased from Biosolve (Westford, MA). CSF Collection. Post-mortem CSF samples from five different patients were collected by ventricular puncture at autopsy, 6 h after death on average. Control ante-mortem CSF samples used for Western-blot and ELISA validation were collected by diagnostic lumbar puncture from five living patients. Characteristics of deceased and living patients have been previously described.17 Each patient or patient’s relatives gave informed consent prior to enrolment. The local institutional ethical committee board approved the clinical protocol. Atraumatic CSF samples were centrifuged immediately after collecting, aliquoted, frozen at -80 °C, and stored until analysis. Depletion of Abundant Proteins. Pooled post-mortem CSF samples were concentrated to 300 µL using 10 kDa MWCO ultrafiltration devices (Vivaspin UF 4, Vivascience, Germany). The protein load was approximately 1.6 mg. The sample was then diluted 1:5 in MARS buffer A (Agilent, Palo Alto, CA) and passed through a 0.22-µm filter. Aliquots of 200 µL were injected on a 4.6 × 100 mm MARS column (Agilent). The flow-through fractions were collected, pooled, and concentrated to approximately 1 mL using ultrafiltration. These concentrated fractions were washed twice with 10 mM NH4HCO3. A protein concentration assay was performed using the Bradford method (Bio-Rad, Hercules, CA).

research articles Off-Gel Electrophoresis. The OGE fractionation was performed as previously described.24 The depleted CSF was prepared for OGE by adding urea, thiourea and DTT to final concentrations of 7 M, 2 M, and 65 mM, respectively. IPG strips (13 cm, pH 4.0-7.0) were rehydrated in a solution containing 7 M urea, 2 M thiourea, 65 mM DTT, 0.5% (v/v) ampholytes (pH 4.0-7.0) and 5% glycerol. A 15-well device was then placed on the rehydrated IPG and 50 µL of sample was loaded in each well across the whole strip. Several multiwell devices were used in parallel to allow fractionation of the whole sample in a single experiment. The voltage was started at 100 V (1 h) then increased to 500 V (for 1 h), 1000 V (for 1 h) and finally to 2000 V where it was maintained for 15 h. The focusing was performed at 20 °C with a current limit of 50 mA. Fractions were recovered from each of the wells. SDS-PAGE and In-Gel Digestion. Proteins from OGE fractions were separated by SDS-PAGE on homemade 12% T TrisGlycine gels (8 × 5 × 0.15 cm). Approximately 60 µL of each fraction was loaded on the gel. After the migration, gels were stained with an MS-compatible silver stain.25 Bands cut from the silver-stained gels were destained with 15 mM K3Fe(CN6), 50 mM Na2S2O3, and washed with Milli-Q water (Millipore, Billerica, MA).26 The gel pieces were then dehydrated in 100% CH3CN and dried in a vacuum centrifuge. The proteins were in-gel digested using standard protocols.27 Peptides were extracted with 1% TFA followed by 50% CH3CN, 0.1% TFA. The combined extracts were concentrated by vacuum centrifugation. LC-MS/MS. Peptides extracted following in-gel digestion were dissolved in 9 µL 5% CH3CN, 0.1% formic acid and 5 µL was loaded for LC-MS/MS analysis. A precolumn (100 µm inner diameter, 2-3.5 cm long) was connected directly to an analytical column (75 µm inner diameter, 9-10 cm long). Both columns were packed in-house with 5 µm, 3 Å Zorbax Extend C-18 (Agilent). A gradient from 4 to 56% solvent B in solvent A (Solvent A: 5% CH3CN, 0.1% formic acid, Solvent B: 80% CH3CN, 0.1% formic acid) was developed over 15 min at a flow rate of approximately 300 nl/min. The concentration of solvent B was increased to 95% before returning to start conditions for re-equilibration of the column. The eluate was sprayed directly into the nanoESI source of an LCQ DecaXP ion trap mass spectrometer (Thermo Finnigan, San Jose, CA) with a spray voltage of 1.8-2.2 kV. Data dependent acquisition was used to automatically select 2 precursors for MS/MS from each MS spectrum (m/z range 400-1600). MS/MS spectra were acquired with a normalized collision energy of 35%, an activation Q of 0.25 and an isolation width of 4 m/z. The activation time was 30 ms. Dynamic exclusion was applied with a repeat count of 2, an exclusion time of 30 s, and an exclusion peak width of (1.5 Da. Wideband activation was also applied. Maximum injection times of 50 ms and 200 ms were used for MS and MS/MS acquisitions, respectively, and the corresponding automatic gain control targets were set to 108. Data Extraction and Database Interrogation. Peak lists were generated using Bioworks 3.1 software (Thermo Finnigan, San Jose, CA). The resulting dta files from each analysis were automatically combined into a single text file. The resulting peak lists were searched against the UniProt/Swiss-Prot database without species restriction using Mascot operating on a local server (version 1.8, Matrix Sciences, U.K.) and Phenyx Virtual Desktop (Gene Bio, Switzerland). Mascot was used with average mass selected, a precursor mass error of 2.0 Da and a peptide mass error of 1.0 Da. Trypsin was selected as the Journal of Proteome Research • Vol. 5, No. 7, 2006 1675

research articles enzyme, with a single potential missed cleavage. ESI ion trap was selected as the instrument type and oxidized methionine as a variable modification. For Phenyx, ion trap was selected for the instrument type and LCQ for the algorithm. Two search rounds were used, both with trypsin selected as the enzyme and oxidized methionine as a variable modification. In the first round, one missed cleavage was allowed and the normal cleavage mode was used. This round was selected in “turbo” search mode. In the second round, two missed cleavages were allowed and the cleavage mode was set to half-cleaved. The minimum peptide length allowed was 6 amino acids and the parent ion tolerance was 2.0 Da in both search rounds. The acceptance criteria were slightly lowered in the second round search (round 1: AC score 7.0, peptide Z-score 7.0, peptide p value 1 E-6; round 2: AC score 7.0, peptide Z-score 6.0, peptide p value 1 E-5). Proteins that were identified as human proteins with 3 or more high-scoring peptides from both Mascot and Phenyx were accepted to be true matches. “High scoring peptides” corresponded to peptides that were above the threshold in Mascot searches (5% probability of false match for each peptide above this score) and above a peptide score of 8.5 for Phenyx searches using the LCQ scoring algorithm. Matches with fewer than 3 peptides were manually validated. Single peptide matches were only included if they were high scoring peptides in the results from both programs and if the data was considered to match the peptide sequence well. The peak lists were also searched against the UniProt combined Swiss-Prot and TrEMBL database restricted to human entries using Phenyx Virtual Desktop (Gene Bio, Switzerland) as previously described. The acceptance criteria were more stringent than for the search of the Swiss-Prot database alone (round 1: AC score 16.0, peptide Z-score 8.0, peptide p value 1 E-7; round 2: AC score 10.0, peptide Z-score 7.0, peptide p value 1 E-6). Immunoblot Analyses of Ante- and Post-mortem CSF Samples. Post-mortem and ante-mortem CSF samples (20 µL) were loaded on homemade 12% T Tris-Glycine gels (8 × 7 × 0.1 cm). The following positive controls were used: 100 ng of recombinant calcyphosine (Scientific Proteins, Switzerland), 100 ng of recombinant ubiquitin fusion degradation protein 1 (UFD1) (Biosite, San Diego, CA), 1 µL of U373 cell line extract for 14-3-3 protein isoform beta, and 5 µL of HeLa cell line extract for glutathione S-transferase P (GST-P). Proteins separated by SDS-PAGE were electroblotted onto a PVDF membrane as described by Towbin et al.28 Membranes were stained with Amido-Black, destained with water and dried. Immunodetection was performed as previously described29 using specific antibodies and BM Chemiluminescence Western Blotting Kit (Roche, Basel, Switzerland). The following antibodies were used: anti-human calcyphosine rabbit polyclonal antibody (Scientific Proteins, Witterswil, Switzerland) diluted 1/1000, anti-human UFD1 mouse Omniclonal antibody (Biosite, San Diego, CA) diluted 1/1000, anti-human 14-3-3 β rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1/500, anti-human GST-P mouse monoclonal antibody (Transduction Laboratories, Lexington, KY) diluted 1/1000. Immunoblot Detection of 14-3-3 Protein γ in OGE Fractions. Five µL of OGE fractions obtained from post-mortem and ante-mortem CSF pools were loaded on homemade 12% T Tris-Glycine gels (8 × 7 × 0.1 cm). Five µL of crude postmortem and ante-mortem CSF pools were used as positive and negative controls, respectively. Proteins separated by 1-DE were 1676

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electroblotted onto a PVDF membrane as described by Towbin et al.28 Membranes were stained with Amido-Black, destained with water and dried. Immunodetection was performed as previously described29 using anti-human 14-3-3 γ rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1/500 and BM Chemiluminescence Western Blotting Kit (Roche, Basel, Switzerland). Sandwich ELISA Detection of GST-P1. As no commercial kit was available for the detection of GST-P1, a homemade ELISA test was developed. Reproducibility tests showed less than 15% coefficient variation. Sandwich ELISA was performed as described by Allard et al20 in 96-well Reacti-Bind NeutrAvidin coated Black Plates (Pierce, Rockford, IL) using biotinconjugated and alkaline phosphatase-conjugated anti-human GST-P1 monoclonal antibodies. A 50-µL portion of CSF samples diluted 2-fold was used. Each sample was assayed in duplicate and distributed randomly on the plate. Calibrator samples, corresponding to recombinant GST-P1 protein (Invitrogen Corporation, Carlsbad, CA), were run on the same plate. Recombinant GST-P1 was diluted to concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.56, and 0 µg/L in the dilution buffer. Calibration curves were analyzed by linear regression in the linear range of the curve. Protein concentrations in CSF samples were calculated from the calibration curve. Statistical analyses were performed using GraphPad Prism software version 4.0 (GraphPad Software Inc, San Diego, CA). A nonparametric Mann-Whitney test was performed to assess the capability of the GST-P1 level to discriminate between antemortem and post-mortem CSF.

Results Abundant Protein Depletion. The analysis of body fluids, such as CSF, poses a challenge in terms of the high dynamic range of protein concentrations. The dominance of particular proteins such as albumin and immunoglobulins results in many proteins of lower abundance remaining undetected by conventional techniques such as 2-DE and mass spectrometry. Therefore, immunodepletion of some of the most abundant CSF proteins (albumin, serotransferrin, IgG, IgA, haptoglobin, and R-1-antitrypsin) was performed in order to improve the coverage of low abundance proteins. To access the results from depletion of abundant proteins, 2-DE of the post-mortem CSF sample was performed before and after immunoaffinity subtraction (data not shown). The gels showed the depletion of the target proteins including strong spots corresponding to albumin fragments and confirmed that the removal of some abundant proteins enabled the detection of spots of lower abundance. These results obtained for the post-mortem CSF sample reproduced perfectly those presented by Maccarrone et al.14 for ante-mortem CSF with similar depletion reproducibility from run to run (data not shown). Off-Gel Electrophoresis. Following immunoaffinity depletion, the post-mortem CSF proteins were fractionated by OGE according to their pI. OGE was performed using a pH gradient ranging from 4.0 to 7.0. The fractions obtained from OGE were then separated by SDS-PAGE. Figure 1 shows a silver-stained SDS-PAGE gel of post-mortem CSF fractions. The quality of the OGE fractionation can be seen from Figure 1, with some bands represented in multiple fractions and others concentrated in one or two fractions. Western blots were also used to verify the quality of the OGE fractionation. In Figure 2, Western blot analysis using a specific antibody for the gamma isoform of the 14-3-3 protein is shown. A sample of the pooled post-

Brain Cell Proteins in Post-mortem CSF

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Figure 1. Silver-stained SDS-PAGE gel of fractions following OGE of the pooled post-mortem samples. CSF proteins were fractionated by OGE as described in the material and methods section. Approximately 60 µL of each OGE fraction was then loaded on homemade 12% T Tris-Glycine gels. Proteins were stained with a MS-compatible silver stain. This image was created from a number of individual SDS-PAGE gels run in different experiments but under identical conditions. The theoretical pH of each OGE fraction is indicated.

Figure 2. Immunoblot analysis of 14-3-3 protein isoform gamma (14-3-3 γ) in OGE fractions from post-mortem CSF (labeled 1-15). OGE fractions (5 µL) were loaded on homemade 12% T Tris-Glycine gels. Crude post-mortem and ante-mortem CSF pools (5 µL) were used as positive and negative controls, respectively. Proteins separated by 1-DE were electroblotted onto a PVDF membrane. Immunodetection was performed as described in the Material and Methods section using an anti-human 14-3-3 γ polyclonal antibody. Arrows highlight the bands that show the presence of 14-3-3 γ in the pooled post-mortem CSF sample and fraction 3 from OGE.

mortem CSF was separated by SDS-PAGE along with each of the fractions from OGE of the sample. As can be seen from Figure 2, 14-3-3 protein gamma was apparent in the unfractionated post-mortem CSF sample and in a single fraction following OGE of the post-mortem CSF sample (fraction 3). These results corresponded with the identifications obtained by MS and database searching. The 14-3-3 protein gamma was identified in one band of fraction 3 from the post-mortem CSF fractionation (see Supporting Information Table 1). The antemortem CSF sample did not show any band for the gamma 14-3-3 protein. Identification by Mass Spectrometry. Proteins were identified from bands cut from the gels shown in Figure 1. A total of 299 proteins were identified in this study and these results are listed in Supporting Information Tables 1 and 2 containing proteins from the UniProt/Swiss-Prot database (searched with all species). Of all the proteins identified, 277 were identified from the Swiss-Prot database and a further 22 from the human TrEMBL searches. Of the 299 proteins that were identified from the post-mortem CSF fractions, 172 were identified for the first time in CSF. Identification Validation by Immunoblot. Proteins of specific interest, such as proteins that were identified for the first time in CSF or those known to be associated with brain disorders, were further investigated using immunoblots. Figure 3 shows Western blots of four proteins that were identified only in post-mortem fractions. As described in the methods section, unfractionated CSF samples were separated on a SDS-PAGE

Figure 3. Immunoblot analyses of 14-3-3 protein isoform beta (14-3-3 β), calcyphosine (CAYP), glutathione S-transferase P (GSTP) and ubiquitin fusion degradation protein 1 (UFD1). postmortem and ante-mortem CSF samples (20 µL) were loaded on homemade 12% T Tris-Glycine gels. Proteins separated by SDSPAGE were electroblotted onto a PVDF membrane. Immunodetection was performed as described in the Material and Methods section using specific antibodies. Lane 1: positive controls (14-3-3 β: 1 µL of U373 cell line extract; CAYP and UFD1: 100 ng of recombinant protein; GSTP: 5 µL of HeLa cell line extract); Lanes 2,4,6,8: ante-mortem CSF samples; Lanes 3,5,7,9: post-mortem CSF samples.

gel and then electroblotted onto a PVDF membrane. The membrane was then probed for proteins of interest using specific antibodies. The results for the 14-3-3 protein beta, Journal of Proteome Research • Vol. 5, No. 7, 2006 1677

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further suggests that the majority of the proteins identified in post-mortem CSF are associated with intracellular functions.

Discussion

Figure 4. GSTP-1 concentrations in ante-mortem (n ) 4) (mean 4.42 µg/L; range, 3.88-5.18 µg/L) and post-mortem (n ) 4) CSF (mean 899.47; range, 671.39-1120.7 µg/L; p ) 0.0286).

Figure 5. (A) Pie charts showing the classification of post-mortem CSF proteins identified from the UniProt Swiss-Prot database according to localization. (B) Pie charts showing the functional classification of post-mortem CSF proteins identified from the UniProt/Swiss-Prot database.

calcyphosine, GST-P, and UFD1 are shown in Figure 3. For the first three proteins, evidence for their increased concentration in post-mortem CSF compared to ante-mortem CSF is clear from the strong signal apparent in each of the post-mortem CSF samples but not in the ante-mortem samples. The result is less clear for UFD1, but an increased concentration of this protein in the post-mortem CSF samples is still apparent. Other isoforms of the 14-3-3 protein were also tested (epsilon, gamma, teta, zeta) and gave results identical to isoform beta (data not shown). ELISA Validation of GST-P1 Differential Expression. To further investigate and validate the presence of a protein (GSTP1) that was identified in the post-mortem CSF sample, a homemade ELISA sandwich was developed. The concentration of GST-P1 was determined in ante- and post-mortem CSF samples. Figure 4 shows a significant increase (p ) 0.0286) of GST-P1 level in the post-mortem CSF (n ) 4) compared to antemortem CSF (n ) 4). Localization and Functional Classification. Bibliographic searches of the proteins identified from the UniProt/Swiss-Prot database enabled their classification by their putative localization and function. The pie-chart in Figure 5A shows most of the proteins identified in the post-mortem CSF sample had a putative intracellular localization (57.5%) and there was a lower proportion of classical circulating proteins (21%) and secreted proteins (3%). These data strongly suggest that most of these proteins arose in post-mortem CSF by tissue leakage. In postmortem CSF, the proportion of proteins classified as protein binding and transport, immunity or inflammation, was low compared to functional classes such as enzymes, structural proteins, and signal transduction proteins (Figure 5B). This 1678

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A previous 2-DE study identified several proteins with increased levels in post-mortem CSF compared to ante-mortem CSF.17 Further validation studies showed the potential interest of some of these proteins as biochemical markers of various neurological disorders.18-23 The goal of the present study was to further characterize the post-mortem CSF proteome in order to identify new proteins associated with brain cell death. To further mine post-mortem CSF proteome, we performed an analysis using a protocol combining several steps of protein fractionation prior to protein identification. These were immunoaffinity depletion of abundant CSF proteins, off-gel electrophoresis, SDS-PAGE and protein identification by LCMS/MS. A total of 299 proteins was identified, of which 172 proteins were not previously described to be present in CSF. For protein depletion, we used a highly specific method based on immunoaffinity subtraction chromatography. This system minimizes the risk of nonspecific protein removal.14,30,31 CSF proteins were further fractionated according to their pI using OGE. The OGE technique has been shown to reliably separate proteins with a resolution up to 0.15 pH units.24,32 Immunodetection of the gamma isoform of the 14-3-3 protein in a single one of the post-mortem fractions following OGE confirmed the very high resolving power of the technique. SDS-PAGE and 2-DE gel analysis of replicate fractionations of post-mortem CSF samples also confirmed the high reproducibility of OGE (data not shown). The fractions obtained from OGE were separated by SDS-PAGE. After silver staining, the gel lanes were sliced in 1 mm gel bands and in-gel protein digestion and peptide extraction were performed. In the final step of the protocol, proteins were identified by LC-ESI-MS/MS analysis using an ion-trap mass spectrometer. Data-dependent LCESI-MS/MS analysis is often considered to be poorly reproducible between replicated data acquisitions.33 This is generally the case for large-scale proteome studies investigating very complex protein samples. In the study presented here, LC-ESIMS/MS analysis was performed on peptides extracted from small SDS-PAGE gel bands. This approach reduced the complexity of the peptide mixture analyzed and lowered the risk of missed protein identifications. Immunoblot experiments were performed in order to check the identity and specificity of selected post-mortem proteins including 14-3-3β, calcyphosine, GST-P1 and UFD1. ELISA also confirmed the presence of GST-P1 in both ante- and post-mortem CSF samples, but with a level 200 times higher in post-mortem CSF samples. The use of post-mortem CSF as a source of new proteins associated with brain cell death was based on the assumption that the global brain necrosis following death results in protein leakage from damaged tissues into CSF, thereby mimicking events associated with brain tissue lesions in various neurological disorders. Accordingly, more than 75% of the 172 proteins newly identified in the post-mortem CSF sample had a putative intracellular location, most likely due to their leakage from damaged brain cells. In addition, most of the proteins identified from post-mortem CSF were found to be associated with intracellular functions (metabolic enzymes, structural proteins, signal transduction proteins and proteins involved in protein synthesis and degradation). Further support for the argument that most of the proteins specifically identified in post-mortem CSF arose from tissue leakage came from the comparison

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of our results with previous studies of CSF from healthy subjects.4-12,14,15 Bibliographic searches of the 172 proteins specifically identified in the present post-mortem CSF study also revealed that a number of them had previously been described as potential markers of brain disorders. For example, H-FABP and DJ-1, which were previously identified in the post-mortem CSF 2-DE study,17 have been validated as early plasmatic markers of stroke.18-20 H-FABP was also shown to be a marker of CJD and other neurodegenerative dementias.21,22 Glial fibrillary acidic protein and creatine kinase BB have been described as markers of various brain damage-related disorders, although their clinical utility has been questioned.34-37 We also identified several isoforms of the 14-3-3 protein, which is a known CSF marker of CJD.38,39 Another interesting finding was the identification in post-mortem CSF of a fragment of the brain spectrin alpha-chain. Spectrin fragments, called spectrin breakdown products (SBPs), are produced in a variety of neurodegenerative conditions by caspase-3 and calpain-mediated proteolysis.40 They are particularly stable and were proposed as CSF markers of traumatic brain injury.41,42 The fragment identified in this study had a molecular weight of approximately 120 kDa, corresponding to a specific SBP produced by caspase-3 proteolysis. More recently, we demonstrated the utility to array post-mortem CSF proteins as a first step toward the discovery of plasmatic markers of ischemic brain injury, through the validation of PARK7 and NDKA.20 Highly significant increases of both biomarker levels were found with a sensitivity of 54 to 91% for PARK7 and of 70 to 90% for NDKA, and a specificity of 80 to 97% for PARK7 and of 90 to 97% for NDKA. The concentration of both biomarkers showed an early elevation, within 3 h of stroke onset. Similarly, highly significant increase of UFD1 was found in Swiss stroke patients with 71% sensitivity (95% CI, 52 to 85.8%), and 90% specificity (95% CI, 74.2 to 98%) (n ) 31, p < 0.0001). Significant elevated concentration of this marker was also found in Spanish (n ) 39, p < 0.0001, 95% sensitivity (95% CI, 82.7 to 99.4%), 76% specificity (95% CI, 56.5 to 89.7%)) and North-American stroke patients (n ) 53, 62% sensitivity (95% CI, 47.9 to 75.2%), 90% specificity (95% CI, 73.5 to 97.9%), p < 0.0001). Its concentration was increased within 3 h of stroke onset, on both the Swiss (p < 0.0001) and Spanish (p ) 0.0004) cohorts (Allard et al., submitted). Many additional protein identifications from this study are of interest as potential markers of brain disorders owing to their elevated levels in post-mortem CSF. From the list of 172 proteins newly identified in post-mortem CSF, several have been previously highlighted since they have been reported to be brain specific, have high expression levels in the brain and/ or have been associated with nervous system injury or pathology.43-87 Taken together these data strongly suggest that the newly identified post-mortem CSF proteins represent highly interesting potential markers of brain damage. According to our model, they were released from damaged cells into CSF following brain tissue necrosis. In addition, they have been reported to be brain specific or have high expression levels in the brain, thereby increasing the chance of being specific markers of brain injury. Furthermore, altered expression levels of several of these proteins have been found in neurological disorders or following nervous system injury. Validation studies using both serum and CSF samples from patients are necessary to determine the utility of these proteins as markers of brain damage. With this

aim, immunoassays are currently under development for several of these proteins.

Acknowledgment. The authors would like to thank Proteome Sciences plc from United Kingdom and Agilent Technologies for their financial support. Supporting Information Available: Supporting Information on protein identification is included in two files named supplementary Table 1.xls and Supporting Information Table 2.doc This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Anderson, N. L.; Anderson, N. G. The human plasma proteome: history, character, and diagnostic prospects. Mol. Cell. Proteomics 2002, 1, 845-867. (2) Davidsson, P.; Sjogren, M. The use of proteomics in biomarker discovery in neurodegenerative diseases. Dis. Markers 2005, 21, 81-92. (3) Blennow, K. CSF biomarkers for mild cognitive impairment. J. Intern. Med. 2004, 256, 224-234. (4) Davidsson, P.; Paulson, L.; Hesse, C.; Blennow, K.; Nilsson, C. L. Proteome studies of human cerebrospinal fluid and brain tissue using a preparative two-dimensional electrophoresis approach prior to mass spectrometry. Proteomics 2001, 1, 444-452. (5) Yuan, X.; Russell, T.; Wood, G.; Desiderio, D. M. Analysis of the human lumbar cerebrospinal fluid proteome. Electrophoresis 2002, 23, 1185-1196. (6) Sickmann, A.; Dormeyer, W.; Wortelkamp, S.; Woitalla, D.; Kuhn, W.; Meyer, H. E. Towards a high-resolution separation of human cerebrospinal fluid. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2002, 771, 167-196. (7) Dumont, D.; Noben, J. P.; Raus, J.; Stinissen, P.; Robben, J. Proteomic analysis of cerebrospinal fluid from multiple sclerosis patients. Proteomics 2004, 4, 2117-2124. (8) Hammack, B. N.; Fung, K. Y.; Hunsucker, S. W.; Duncan, M. W.; Burgoon, M. P.; Owens, G. P.; Gilden, D. H. Proteomic analysis of multiple sclerosis cerebrospinal fluid. Mult. Scler. 2004, 10, 245-260. (9) Finehout, E. J.; Franck, Z.; Lee, K. H. Towards two-dimensional electrophoresis mapping of the cerebrospinal fluid proteome from a single individual. Electrophoresis 2004, 25, 2564-2575. (10) Yuan, X.; Desiderio, D. M. Proteomics analysis of prefractionated human lumbar cerebrospinal fluid. Proteomics 2005, 5, 541-550. (11) Davidsson, P.; Folkesson, S.; Christiansson, M.; Lindbjer, M.; Dellheden B, Blennow, K.; Westman-Brinkmalm, A. Identification of proteins in human cerebrospinal fluid using liquid-phase isoelectric focusing as a prefractionation step followed by twodimensional gel electrophoresis and matrix-assisted laser desorption/ionisation mass spectrometry. Rapid Commun. Mass Spectrom. 2002, 16, 2083-2088. (12) Hansson, S. F.; Puchades, M.; Blennow, K.; Sjogren, M.; Davidsson, P. Validation of a prefractionation method followed by two-dimensional electrophoresis-Applied to cerebrospinal fluid proteins from frontotemporal dementia patients. Proteome Sci. 2004, 2, 7. (13) Wenner, B. R.; Lovell, M. A.; Lynn, B. C. Proteomic analysis of human ventricular cerebrospinal fluid from neurologically normal, elderly subjects using two-dimensional LC-MS/MS. J. Proteome Res. 2004, 3, 97-103. (14) Maccarrone, G.; Milfay, D.; Birg, I.; Rosenhagen, M.; Holsboer, F.; Grimm, R.; Bailey, J.; Zolotarjova, N.; Turck, C. W. Mining the human cerebrospinal fluid proteome by immunodepletion and shotgun mass spectrometry. Electrophoresis 2004, 25, 2402-2412. (15) Zhang, J.; Goodlett, D. R.; Peskind, E. R.; Quinn, J. F.; Zhou, Y.; Wang, Q.; Pan, C.; Yi, E.; Eng, J.; Aebersold, R. H.; Montine, T. J. Quantitative proteomic analysis of age-related changes in human cerebrospinal fluid. Neurobiol. Aging 2005, 26, 207-227. (16) Zhang, J.; Goodlett, D. R.; Quinn, J. F.; Peskind, E.; Kaye, J. A.; Zhou, Y.; Pan, C.; Yi, E.; Eng, J.; Wang, Q.; Aebersold, R. H.; Montine, T. J. Quantitative proteomics of cerebrospinal fluid from patients with Alzheimer disease. J. Alzheimers Dis. 2005, 7, 125313.

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