Use of Peptide Analogue Diversity Library Beads for Increased Depth

Charleston, South Carolina 29425, and Department of Internal Medicine, University of Texas Southwestern. Medical Center, Dallas, Texas 75390. Received...
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Use of Peptide Analogue Diversity Library Beads for Increased Depth of Proteomic Analysis: Application to Cerebrospinal Fluid Kevin S. Shores,† D. Gomika Udugamasooriya,‡ Thomas Kodadek,‡ and Daniel R. Knapp*,† Department of Pharmacology and MUSC Proteomics Center, Medical University of South Carolina, Charleston, South Carolina 29425, and Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390 Received October 23, 2007

Biological samples can contain proteins with concentrations that span more than 10 orders of magnitude. Given the limited dynamic range of analysis methods, observation of proteins present at the lower concentrations requires depletion of high-abundance proteins, or other means of reducing the dynamic range of concentrations. Hexapeptide diversity library beads have been used to bind proteins in a complex sample up to a given saturation limit, effectively truncating the maximum concentration of proteins at a desired level. To avoid the potential problem of susceptibility of the hexapeptides to cleavage by proteases in the sample and/or bacterial degradation, peptide analogues that exhibit similar binding characteristics to peptides can be used in place of peptides. We report here the use of hexameric peptoid diversity library beads to reduce the dynamic range of protein concentrations in human cerebrospinal fluid (CSF). Using this method in conjunction with 2D LC/MS/MS analyses, we identified 200 unique proteins, about twice the number identified in untreated CSF. Keywords: peptoid diversity library • cerebrospinal fluid • LC/MS/MS

1. Introduction Identification of disease biomarkers within complex protein samples is challenging, since biomarkers are generally expected to be present in far lower abundance than the proteins that constitute the bulk of the sample. Since proteomics analysis methods have a limited dynamic range, the presence of highabundance species can preclude observation of low-abundance proteins. To facilitate a greater depth of analysis to observe low-abundance species, it is necessary to remove highabundance proteins from the sample prior to analysis. A variety of methods have been explored for removal of high-abundance proteins from complex protein samples, with antibody-based methods being most extensively applied. Whereas most methods have involved specific removal of predefined highabundance species, a new approach is based upon a general reduction in the maximum concentration of all proteins, thereby shifting the observed dynamic range to lower concentrations.1 We report here a new variation of this approach with demonstration of its use for CSF proteomic analysis. The most common sample for human biomarker research has been serum, a complex sample containing proteins with concentrations that span over 12 orders of magnitude.2 Cerebrospinal fluid (CSF) is similar to human serum in terms of dynamic range of protein concentration; however, the overall protein content of CSF is less than 0.5% that of serum.3 Similar to serum, however, CSF contains proteins with concentrations * To whom correspondence should be addressed. E-mail: knappdr@ musc.edu. † Medical University of South Carolina. ‡ University of Texas Southwestern Medical Center.

1922 Journal of Proteome Research 2008, 7, 1922–1931 Published on Web 03/22/2008

that span at least 10 orders of magnitude, with several of the high-abundance CSF proteins originating from human serum. Human CSF is of particular interest due its high turnover rate (7–8 h)3 and its continuous contact with the ventricles of the brain and spinal cord. Consequently, proteomic analysis of CSF provides an appealing approach to monitor the state of the central nervous system (CNS). Several methods of removing high-abundance serum proteins have been used to process CSF with varying levels of effectiveness. Cibacron Blue chromatography has been commonly used to remove albumin from serum; however, it is a nonspecific method and has the potential to also bind proteins of interest and has not often been used to process CSF.4 Solvent depletion methods using various concentrations of acetonitrile have been used to process CSF by Zhang et al.5 to selectively fractionate human serum albumin (HSA), immunoglobulins, and lower abundance proteins. Subsequent proteomic analysis of all three fractions has enabled the identification of more than 300 proteins (although as little as one peptide sequence was used in the identification of a protein). Noben et al. have enabled the identification of 148 proteins in CSF by first employing a 50 kDa molecular weight cutoff filter to remove albumin (∼66 kDa) and other high molecular weight proteins prior to analysis.6 In recent years, serum immunodepletion products have been used to deplete CSF, since CSF contains high levels of several serum proteins, including albumin, immunoglobulins, antitrypsin, and haptoglobin. Maccarrone et al. successfully identified 100 proteins in CSF by processing a pooled CSF sample with the Multiple Affinity Removal System (MARS, Agilent Technologies),7 which includes an HPLC col10.1021/pr7006889 CCC: $40.75

 2008 American Chemical Society

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Use of Peptide Analogue Diversity Library Beads umn that binds HSA, transferrin, IgG, IgA, antitrypsin, and haptoglobin, prior to 2D LC/MS analysis. Ogata et al. compared the performance of the MARS HPLC column to that of a similar HPLC column that contains avian IgY antibodies to HSA, transferrin, IgG, IgA, IgM, and fibrinogen (ProteomeLab, Beckman Coulter) and noted the superior performance of the MARS column, likely owing to the removal of antitrypsin and haptoglobin which are highly abundant in CSF.8 Although successful at removing several high-abundance proteins from CSF, recent evidence suggests that serum immunodepletion methods are only moderately effective in facilitating a deeper analysis of the CSF proteome.9 Since CSF contains several other highabundance proteins that are not targeted by these methods, including prostaglandin D2 synthase, cystatin C, and transthyretin,3 peptides from these proteins dominate the ion current during mass spectrometric analysis. Therefore, to lower the range of observable protein concentrations, depletion of all high-abundance proteins is required.9 Righetti et al. have recently demonstrated an abundant protein depletion method where the maximum concentration of all proteins in a complex mixture is reduced.1,10 This is done by exposing a complex protein sample to a hexapeptide combinatorial library on beads. Individual proteins in the complex sample bind to their respective highest affinity ligands up to the capacity of the aliquot of beads used. The diversity of peptide ligands present in the library (theoretically greater than 106) is far greater than the number of different proteins in a complex sample, which makes it highly probable that there will be one or more high-affinity ligands present for any protein in the sample.1 High-abundance proteins bind to their saturation limit, and amounts in excess of the limit pass through the beads to waste. Lower abundance proteins present in amounts below the saturation limit are extensively bound. After washing the unbound proteins, the bound species are eluted yielding a sample mixture whose concentration range has been truncated at the saturation level. Through binding every protein in a complex sample up to a saturation limit, the overall dynamic range of protein concentrations in a complex sample is effectively lowered. A potential problem of processing raw biological samples with peptide ligand libraries is the likelihood of proteases present in the sample, which could degrade the peptide ligands and alter the binding characteristics of the library. The terminal residue in the hexapeptide libraries is attached as the D-amino acid to reduce this possibility, but the peptides could still be susceptible to internal cleavage. The hexapeptide library would also be expected to be subject to bacterial degradation which complicates storage. The susceptibility of the peptide ligands to proteases or bacterial degradation can be avoided by using peptoids,11–13 peptide analogues with the side chains attached to amino nitrogens rather than the alpha carbons. Peptoids exhibit similar binding characteristics as peptide ligands, but are not subceptible to cleavage by proteins. We demonstrate here the fast and effective use of hexameric peptoid ligand library beads in the depletion of high-abundance proteins from CSF. The comparison of results from the 2D LC/MS/MS analysis of a pooled and depleted CSF sample versus analysis of the same sample prior to depletion has indicated a substantial reduction of all high-abundance protein concentrations and resulted in the identification of 200 unique proteins.

2. Experimental Procedures 2.1. Materials. Amicon Ultra 5 kDa molecular weight cutoff filters were obtained from the Millipore Corporation (Billerca, MA), centrifuge columns (1.8 mL) were obtained from Pierce Biotechnology (Rockford, IL), and 2.0 mL C-18 Sep-Pak vacuum manifold solid phase extraction cartridges (SPE) were obtained from the Waters corporation (Milford, MA). Urea, thiourea, CHAPS, dithiothreitol, iodoacetamide, proteomics grade trypsin, and a bicinchoninic acid (BCA) kit for total protein determination were obtained from Sigma-Aldrich corporation (St. Louis, MO). NovaSyn TG amino resin was obtained from EMD biosciences (San Diego, CA). Allylamine, piperonylamine, furfurylamine, and boc-diaminobutane were obtained from Acros Organics (Pittsburgh, PA), (R)-methylbenzylamine and methoxyethylamine were obtained from Sigma-Aldrich corporation, isobutylamine was obtained from Alfa Aesar (Ward Hill, MA), and ethanolamine was obtained from CSPS Pharmaceuticals, Inc. (San Diego, CA). 2.2. Human CSF. A pool of human CSF was generated by combining multiple deidentified excess clinical specimens obtained under institutional review board approval from the MUSC Hospital clinical chemistry laboratory. A 6.3 mL aliquot was centrifuged at 3000g for 30 min, and two aliquots (5.0 mL for abundant protein depletion and 1.0 mL for control) were removed and isolated. A 100 µL aliquot of CSF was also set aside for total protein determination using a BCA protein assay. The total protein concentration was determined to be 454 µg/ mL. 2.3. Buffer Exchange and Removal of Biological Salts. The 5.0 and 1.0 mL aliquots of CSF were loaded into separate 15.0 mL capacity 5 kDa Amicon Ultra molecular weight cutoff filters. The volume was then increased to 15.0 mL total by adding 0.2 M NH4HCO3, and the cartridges were centrifuged at 3000g until the volume was reduced to 200 µL. This buffer exchange process was repeated two more times, and the retentates from both samples were then dried using vacuum centrifugation. 2.4. Preparation of Hexapeptoid Combinatorial Solid Phase Library Beads. The syntheses of the hexapeptoid library were performed in 8 standard 25 mL glass peptide synthesis reaction vessels (Chemglass, Inc., Vineland, NJ). NovaSyn TG resin (1.0 g; 90 µm; substitution: 0.20–0.30 mmol/g resin; 1.0 g ) 2.9 × 106 beads) was distributed equally into the 8 reaction vessels, 5.0 mL of DMF was added to each vessel, and the beads were allowed to swell at room temperature for 60 min. The DMF was drained, 1.0 mL of 2.0 M bromoacetic acid and 1.0 mL of 3.2 M diisopropylcarbodiimide (DIC) were added to each vessel, and the vessels were gently agitated for 30 s. The vessels were then placed in a beaker inside the center of a 1000 W microwave oven, and 10% power was delivered for 15 s. The vessels were then agitated for 15 s followed by a second 10% power delivery for 15 s. The solution was then drained from each vessel, and each bead aliquot was thoroughly washed with DMF (8 × 2.0 mL) followed by anhydrous DMF (2 × 2.0 mL). The beads in each vessel were treated with 2.0 mL of one of eight primary amines (2.0 M in DMF, Figure 1), the vessels were placed in a beaker in the center of the microwave oven, and 10% power was delivered for 15 s. The vessels were then agitated for 15 s followed by a second 10% power delivery for 15 s. The solution was then drained from each vessel, and each bead aliquot was thoroughly washed with DMF (8 × 2.0 mL). The beads in each vessel were transferred into a single 250 mL synthesis vessel, the solution was drained, the beads were Journal of Proteome Research • Vol. 7, No. 5, 2008 1923

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Figure 1. The amines employed to make the peptoid library. The italicized nitrogen becomes the main chain nitrogen in the peptoid.

suspended in 50 mL of dichloromethane/DMF (2:1), and the beads were randomized through gentle agitation. The beads were then redistributed equally into eight 25 mL peptide synthesis vessels. This process was repeated five additional times. At the completion of the library synthesis, the beads in each vessel were washed thoroughly with DMF (8 × 2.0 mL) followed by dichloromethane (3 × 3.0 mL). The solutions were drained, and the beads were treated with 5.0 mL of TFA/triisopropylsilane/water (95:2.5:2.5) for 2 h to remove side chain protection groups. The solution was drained, and the beads were washed

Shores et al. thoroughly with dichloromethane (8 × 3.0 mL). The beads were then neutralized by treatment with 10% diisopropylethylamine in DMF for 5.0 min and washed with dichloromethane (5 × 3.0 mL). The beads in each reaction vessel were then combined and dried. 2.5. Abundant Protein Depletion. Peptoid beads (hexapeptoid combinatorial solid phase library on beads, 0.1 g) were loaded into two 1.8 mL capacity centrifuge columns, 650 µL of 25 mM NaH2PO4 was added to each column, and the columns were gently agitated for 60 s. The CSF proteins sample was solubilized in 1.4 mL of 25 mM NaH2PO4, and 700 µL of solubilized protein sample was loaded into two separate columns. The columns were then gently agitated for 30 min at room temperature to ensure complete binding to the hexapeptoid library. The stopper on the bottom of the centrifuge columns was then removed, and the columns were placed into 2.0 mL conical collection tubes. The tubes were centrifuged, and the unbound proteins were collected and discarded. The beads were washed with 500 µL of 25 mM NaH2PO4, and the flowthrough was discarded. Bound proteins were eluted first with 500 µL of 2.2 M thiourea, 7.7 M urea, and 4.4% CHAPS, followed by 500 µL of 9.0 M urea, pH 3.8 (5% (v/v) acetic acid). Protein elution fractions from both columns were pooled and loaded into a 15.0 mL capacity 5 kDa molecular weight cutoff filter. A buffer exchange was performed with NH4HCO3 as described in section 2.3. The retentate (200 µL) was then removed from the filter, and a 10 µL aliquot was set aside for total protein determination. The total protein content was determined to be 100 µg, which indicated a 95.6% reduction in total protein content of the original CSF. 2.6. Reduction, Akylation, and Digestion of CSF Proteins. CSF proteins from both the depleted and control sample were denatured in 450 µL of 8.0 M urea. Disulfide bonds were

Figure 2. Unique proteins identified following consecutive shotgun analyses of the control CSF sample (bottom) and the peptoid depleted sample (top line). 1924

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Table 1. Proteins Identified in the Control CSF Sample protein

Mr

Uniprot ID

TSC

% abun.

albumin hypothetical protein LOC651928 cystatin C transthyretin hypothetical protein LOC649897 transferrin prostaglandin D2-synthase alpha-1-antitrypsin apolipoprotein A-I preproprotein apolipoprotein E apolipoprotein A-II preproprotein anti-RhD monoclonal T125 gamma1 heavy chain beta globin haptoglobin-related protein alpha 2 globin beta-2-microglobulin vitamin D-binding protein haptoglobin orosomucoid 1 orosomucoid 2 PREDICTED: similar to Ig alpha-1 chain C region apolipoprotein D serpin peptidase inhibitor, clade A, member 3 complement component 3 hemopexin PREDICTED: similar to Ig kappa chain V-III region clusterin isoform 1 alpha-2-macroglobulin delta globin angiotensinogen preproprotein apolipoprotein A-IV complement component 4A preproprotein chromogranin A alpha-2-glycoprotein 1, zinc dickkopf homologue 3 complement component 1 inhibitor PREDICTED: similar to Ig gamma-4 chain C region retinol-binding protein 4, plasma apolipoprotein H ceruloplasmin (ferroxidase) A-gamma globin serine (or cysteine) proteinase inhibitor, clade F thymosin-like 3 PREDICTED: similar to Ig gamma-3 chain C region PREDICTED: similar to Ig gamma-1 chain C region CD59 antigen p18–20 kallikrein 6 isoform B alpha-2-HS-glycoprotein CD14 antigen serine (or cysteine) proteinase inhibitor, clade C gelsolin isoform B epididymal secretory protein E1 alpha 1B-glycoprotein proprotein convertase subtilisin/kexin type 1 inhibitor kininogen 1 secreted phosphoprotein 1 isoform A complement factor B preproprotein VGF nerve growth factor inducible alpha-1-microglobulin/bikunin complement component 1, q subcomponent, B chain amyloid precursor-like protein 1 isoform 2 carnosinase 1 complement component 1, q subcomponent chitotriosidase osteoglycin preproprotein pancreatic ribonuclease

69321.63 26229.18 15789.08 15877.05 22058.92 76999.66 21015.35 46707.09 30758.94 36131.79 11167.9 52253.29 15988.29 39004.7 15247.92 13705.91 52883 45176.59 23496.77 23587.64 28490.23 21261.77 47620.63 187045.3 51643.32 16578.49 57795.68 163188.3 16045.29 53120.61 45344.51 192663.6 50657.71 34237.13 38365.15 55119.49 47414.69 22995.26 38286.68 122127.6 16118.27 46283.37 5059.536 18069.91 64280.8 14167.79 15045.3 39299.73 40050.79 52568.98 80590.58 16559.49 54219.66 27355.79 47852.68 35401.25 85478.58 67217.77 38973.99 26704.49 72131.33 56656.2 25757.13 51648.38 33900.89 17632.68

P02768 946295 P01034 P02766 Q49AS2 P02787 Q5SQ09 P01009 P02647 P02649 P02652 Q5EFE5 P68871 P00739 Q86YQ5 Q9UM88 P02774 P00738 P02763 Q5T538 Q8NCL6 P05090 P36955 Q6LDJ0 P02790 642113 Q96AJ1 P01023 P02042 P01019 P06727 Q5JNX2 P10645 P02765 Q4R417 060860 P01861 P02753 P02749 P00450 Q14474 Q4R6H4 P11885 P01860 652050 107271 Q92876 P02765 P08571 P32261 137150 P61916 P04217 P29120 P01042 000571 P00751 Q9UDW8 Q5TBD7 Q5T960 Q5T012 Q96KN2 Q5T960 Q13231 P20774 P07998

1830 165 85 73 101 314 79 160 88 72 22 100 30 64 25 21 73 61 28 28 33 21 44 172 42 12 37 103 10 32 26 101 26 17 19 27 23 11 18 55 7 20 2 7 24 5 5 13 13 17 26 5 16 8 13 9 21 16 9 6 15 11 5 10 6 3

25.62 6.10 5.22 4.46 4.44 3.96 3.65 3.32 2.78 1.93 1.91 1.86 1.82 1.59 1.59 1.49 1.34 1.31 1.16 1.15 1.12 0.96 0.90 0.89 0.79 0.70 0.62 0.61 0.60 0.58 0.56 0.51 0.50 0.48 0.48 0.48 0.47 0.46 0.46 0.44 0.42 0.42 0.38 0.38 0.36 0.34 0.32 0.32 0.31 0.31 0.31 0.29 0.29 0.28 0.26 0.25 0.24 0.23 0.22 0.22 0.20 0.19 0.19 0.19 0.17 0.17

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Table 1. Continued

a

protein

Mr

Uniprot ID

TSC

% abun.

limbic system-associated membrane protein complement factor H isoform B secretogranin III prion protein preproprotein histidine-rich glycoprotein galectin 3 binding protein complement factor H isoform A lysozyme insulin-like growth factor binding protein 2, 36k complement component 1, r subcomponent Thy-1 cell surface antigen vitronectin plasminogen fibrinogen, beta chain preproprotein coagulation factor II SPARC-like 1 fibronectin 1 isoform 3 preproprotein C-type lectin domain family 3, member B family with sequence similarity 3, member C autotaxin isoform 2 preproprotein chromogranin B complement component 7 Chitinase 3-like 1 prosaposin insulin-like growth factor binding protein 7 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltra afamin alpha-2-plasmin inhibitor PREDICTED: similar to Ig gamma-2 chain C region fibulin 1 isoform B cell adhesion molecule with homology to L1CAM fibrinogen, alpha polypeptide isoform alpha fibulin 1 isoform C fibrinogen, gamma chain isoform gamma-B precursor neuronal cell adhesion molecule isoform B EGF-containing fibulin-like extracellular matrix calsyntenin 1 isoform 1 complement component 9 brevican isoform 1 peptidoglycan recognition protein L complement component 1, s subcomponent complement component 4B preproprotein fibulin 1 isoform D gelsolin isoform A neural cell adhesion molecule 1 isoform 1 interalpha (globulin) inhibitor H4 interalpha globulin inhibitor H2 polypeptide insulin receptor substrate 1 Fc fragment of IgG binding protein

37370.23 50974.1 52972.91 27643.19 59540.94 65289.4 138978.4 16526.29 35114.41 80147.95 17923.35 54271.23 90510.23 55892.23 69992.2 75201.31 259061 22552.29 24664.59 98929.59 78199.14 93457.27 42586.39 58073.93 29111.45 47088.94 69024.09 54561.16 39968.81 65427.26 136612.7 69713.77 74412.02 51478.88 130964.4 54604.29 109723.7 63132.78 99056.16 67927.6 76634.85 192629.6 77190.46 85644.25 93302.63 103293.2 106396.8 131509 571719.8

Q13449 P08603 Q8WXD2 P04156 P04196 P17931 P08603 Q8N1E2 P18065 Q53HT9 Q59GA0 P04004 P06733 P02675 Q53H04 Q14515 P02751 P05452 Q5HY75 1035181 P05060 Q8TCS7 P36222 O75905 Q16270 O43505 P43652 P08697 P01859 Q59G97 Q59FY0 P02671 Q8N9G0 068656 A4D0S3 Q12805 O94985 Q9UGI4 Q59F90 P19827 Q53HU9 Q53HU9 Q96K89 A2A418 P13592 Q59FS1 A2RTY6 Q9UQB8 P01876

6 8 8 4 8 8 17 2 4 9 2 6 10 6 7 7 24 2 2 8 6 7 3 4 2 3 4 3 2 3 6 3 3 2 5 2 4 2 3 2 2 5 2 2 2 2 2 2 2

0.16 0.15 0.15 0.14 0.13 0.12 0.12 0.12 0.11 0.11 0.11 0.11 0.11 0.10 0.10 0.09 0.09 0.09 0.08 0.08 0.07 0.07 0.07 0.07 0.07 0.06 0.06 0.05 0.05 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.03 0.03 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.01 0.00

Relative abundance was calculated by normalizing the total spectrum count (TSC) of a protein by its molecular weight (Mr).

reduced in 10 mM dithiothreitol for 1 h at 37 °C in the dark with constant agitation, and cysteines were alkylated in 50 mM iodoacetamide for 1 h at 37 °C in the dark with constant agitation. The solution was then diluted to 1.85 M urea with addition of 0.2 M NH4HCO3 (pH 8.5). Trypsin was added (20 µg), and the proteins were digested overnight at 37 °C in the dark with constant agitation. Tryptic peptides were then extracted using a C-18 Sep-Pak Vac solid phase extraction cartridge (Waters Corporation). Peptides were eluted with 70% acetonitrile/0.1% formic acid and then dried using vacuum centrifugation. 2.7. 2D LC/MS/MS. CSF peptides from both samples were processed using a previously reported 2D LC/MS/MS method.9 1926

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Briefly, offline strong cation exchange (SCX) chromatography was used to separate each CSF peptide sample. A 1 h linear potassium chloride (KCl) gradient (0–0.5 M) was applied to each sample, and eight fractions were collected (5 min intervals). The SCX fractions where then separated by reversed-phase (RP) nano-LC, and peptides were analyzed online with a LTQ mass spectrometer (Thermo Electron) with a nanospray ion source. The mass spectrometer was programmed to perform MS/MS analysis on the 5 most intense ions observed in the MS spectrum collected over m/z 400–2000. Four replicate RP LC/ MS/MS analyses were performed for each set of eight SCX fractions from the depleted sample, and three RP LC/MS/MS analyses were performed for each set of eight SCX fractions

Use of Peptide Analogue Diversity Library Beads

research articles the efficiency of the depletion process. Most of the abundant proteins in the depleted sample are also of high abundance in the control sample, including alpha-1-antitrypsin, albumin, transthyretin, prostaglandin D2-synthase, and cystatin C; however, their relative abundances are slightly different. For example, alpha-1-antitrypsin is the most abundant protein in the depleted sample (8.58%), while it is the eighth most abundant protein in the control sample (3.23%). Contrary to this, Cystatin C is less abundant in the depleted sample (2.41%) than in the control sample (5.22%). This variation in abundance is likely due to the binding efficiency of the peptoid library. Despite this variability, however, a general leveling of the highabundance proteins is observed, as the relative abundance of all of the high-abundance proteins in the depleted sample lies between 1 and 8.5% composition (Table 2).

Figure 3. Protein identified in each of the four shotgun analyses of the depleted CSF sample.

from the control sample. All MS/MS spectra were analyzed using Bioworks 3.2 (Thermo Electron) and were searched against an indexed Homo sapiens database, which was extracted from the NCBI nonredundant H. sapiens database, using the Turbo SEQUEST algorithm.14 With the use of Bioworks 3.2, multiconsensus reports were generated for each set of eight RP analyses (four data sets for the depleted sample and three data sets for the control sample). Confident protein identifications in each data set were generated by applying protein and peptide filters, as discussed previously.9

3. Results and Discussion The undepleted (control) CSF protein sample was analyzed by 2D LC/MS/MS. Three consecutive sets of RP LC/MS/MS analyses resulted in the identification of 86, 94, and 85 unique proteins, for combined totals of 108 and 115 unique proteins after two and three runs, respectively (Figure 2). Since the total proteins identified were already approaching a plateau after three runs, a fourth run was not made for the control sample. Table 1 (Table S1 in Supporting Information) was generated by combining the proteins identified in each experiment. The total number of peptide MS/MS spectra that were matched to a particular protein was tabulated for each protein identification (total spectrum count, TSC).15 Proteins were ranked according to their relative abundance, which was calculated by normalizing the TSC of each protein by its molecular weight (Mr). Following depletion with the peptoid beads, the CSF protein sample was analyzed using the same 2D LC/MS/MS method as used for the undepleted sample. Four consecutive analyses resulted in the identification of 142, 148, 157, and 156 unique proteins (Figure 3), for a total of 200 unique proteins. The proteins identified in each analysis were combined in the same manner as in the control sample and were tabulated according to their relative abundance (Table 2). Figure 3 displays the number of proteins identified in each analysis and the overlap of the protein identifications with the other analyses. Of the 200 unique proteins, more than half (108) were identified in each analysis, while 42 unique proteins were identified in only a single LC/MS/MS analysis. Additional consecutive analyses beyond four were not performed, since they would not be expected to generate a significant increase in protein identifications.16 A comparison of abundant proteins identified in the undepleted versus the depleted samples (Tables 1 and 2) illustrates

In a previous study, we compared the performance of three methods of depleting high-abundance proteins in CSF, including two immunodepletion methods (MARS, Agilent Technologies and Proteoseek, Pierce Biotechnology) and ultrafiltration using a 50 kDa molecular weight cutoff filter.9 Following LC/ MS/MS analysis, the two immunodepletion methods were found to be superior to ultrafiltration and facilitated the identification of 171 (MARS) and 163 (Proteoseek) proteins, while 135 proteins were identified in the control, which constituted approximately 27% and 21% improvement, respectively, over analysis without depletion. In the present study, depletion with the peptoid bead library facilitated identification of an average of 151 proteins per analysis, while an average of 88 proteins per analysis were identified in the control, or approximately 58% improvement over analysis without depletion. In the previous study, the immunodepletion methods proved to be very effective in removing their target proteins; however, even in the case where six of the high-abundance proteins were removed (MARS column), the presence of many additional high-abundance proteins limited the depth of the analysis. In comparing the two studies, it should be noted that the 20 most abundant proteins identified in the control sample in the present study (Table 1) were also seen in high abundance in CSF pool analyzed in the previous study. In the present study, the general depletion or “leveling” of high-abundance proteins with the peptoid diversity library allowed detection of a greater number of lower abundance proteins than with immunodepletion in the previous study. Further comparison of the efficiencies of the depletion methods used in the studies on the basis of identification of low-abundance proteins is not possible, since different CSF pools were used in the two studies. While a direct comparison of total spectrum count (TSC) for proteins identified in the control sample versus the depleted sample is not possible (since only three analyses were run for the control sample while four analyses were run for the depleted sample), it is possible to compare the TSC associated with proteins above 1% abundance in each sample. In the undepleted sample, the TSC for proteins above 1% abundance (21 proteins) is 3542 or 72.2% of the TSC for all proteins identified in the undepleted sample (Table 1). In the depleted sample, however, the TSC for proteins above 1% abundance (18 proteins) is 3196 or 48.8% of the TSC for all proteins identified in the depleted sample (Table 2). Clearly, more time is spent analyzing peptides from high-abundance proteins in the undepleted sample versus that in the depleted sample. As a result, approximately twice as many unique proteins were identified in a single analysis of the depleted sample (142, 148, 157, and 156) compared to that in the control sample (86, 94, Journal of Proteome Research • Vol. 7, No. 5, 2008 1927

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Table 2. Proteins Identified in the Peptoid Depleted CSF Sample

1928

protein

Mr

Uniprot ID

TSC

% abun.

alpha-1-antitrypsin albumin hypothetical protein LOC651928 transthyretin apolipoprotein A-I preproprotein apolipoprotein A-II preproprotein hypothetical protein LOC649897 apolipoprotein E alpha 2 globin beta globin cystatin C complement component 3 anti-RhD monoclonal T125 gamma1 heavy chain Prostaglandin D2-synthase complement component 4A preproprotein A-gamma globin PREDICTED: similar to Ig alpha-1 chain C region angiotensinogen preproprotein transferrin * calmodulin 3 clusterin isoform 1 apolipoprotein A-IV hemopexin delta globin chromogranin A dickkopf homologue 3 * apolipoprotein C-III serine (or cysteine) proteinase inhibitor, clade F CD14 antigen orosomucoid 2 alpha-2-macroglobulin carnosinase 1 ceruloplasmin (ferroxidase) complement component 1, q subcomponent, B chain * complement factor D preproprotein complement component 1 inhibitor proprotein convertase subtilisin/kexin type 1 inhibitor osteoglycin preproprotein serpin peptidase inhibitor, clade A, member 3 galectin 3 binding protein gelsolin isoform B coagulation factor II complement factor H isoform B apolipoprotein D serine (or cysteine) proteinase inhibitor, clade C PREDICTED: similar to Ig kappa chain V-III region * serine (or cysteine) proteinase inhibitor, clade A SPARC-like 1 vitamin D-binding protein complement component 1, r subcomponent complement factor B preproprotein histidine-rich glycoprotein plasminogen complement factor H isoform A * paraoxonase 1 Chitinase 3-like 1 * cutA divalent cation tolerance homologue isoform alpha-2-plasmin inhibitor orosomucoid 1 * secreted protein, acidic, cysteine-rich (osteonectin) haptoglobin-related protein alpha 1B-glycoprotein peptidoglycan recognition protein L secretogranin III * Crystallin, alpha A insulin-like growth factor binding protein 7

46707.09 69321.63 26229.18 15877.05 30758.94 11167.9 22058.92 36131.79 15247.92 15988.29 15789.08 187045.3 52253.29 21015.35 192663.6 16118.27 28490.23 53120.61 76999.66 16826.84 57795.68 45344.51 51643.32 16045.29 50657.71 38365.15 10845.5 46283.37 40050.79 23587.64 163188.3 56656.2 122127.6 26704.49 27015.87 55119.49 27355.79 33900.89 47620.63 65289.4 80590.58 69992.2 50974.1 21261.77 52568.98 16578.49 48511.23 75201.31 52883 80147.95 85478.58 59540.94 90510.23 138978.4 39706.25 42586.39 20911.86 54561.16 23496.77 34609.72 39004.7 54219.66 67927.6 52972.91 19896.9 29111.45

P01009 P02768 946295 P02766 P02647 P02652 Q49AS2 P02649 Q86YQ5 P68871 P01034 Q6LDJ0 Q5EFE5 Q5SQ09 Q5JNX2 Q14474 Q8NCL6 P01019 P02787 P27482 Q96AJ1 P06727 P02790 P02042 P10645 Q4R417 P02656 Q4R6H4 P08571 Q5T538 P01023 Q96KN2 P00450 Q5T960 P00746 060860 P29120 P20774 P36955 P17931 137150 Q53H04 P08603 P05090 P32261 642113 Q86U78 Q14515 P02774 Q53HT9 P00751 P04196 P06733 P08603 P27169 P36222 O60888 P08697 P02763 Q4R5R0 P00739 P04217 Q96PD5 Q8WXD2 P02489 Q16270

558 588 195 112 211 66 106 156 55 55 53 472 110 32 287 24 41 75 103 22 74 58 66 20 62 46 13 54 43 25 159 54 111 23 23 43 21 26 36 48 58 48 33 13 32 10 29 44 30 45 46 32 48 73 20 21 10 26 11 16 18 25 31 24 9 13

8.58 6.09 5.34 5.06 4.92 4.24 3.45 3.10 2.59 2.47 2.41 1.81 1.51 1.09 1.07 1.07 1.03 1.01 0.96 0.94 0.92 0.92 0.92 0.89 0.88 0.86 0.86 0.84 0.77 0.76 0.70 0.68 0.65 0.62 0.61 0.56 0.55 0.55 0.54 0.53 0.52 0.49 0.46 0.44 0.44 0.43 0.43 0.42 0.41 0.40 0.39 0.39 0.38 0.38 0.36 0.35 0.34 0.34 0.34 0.33 0.33 0.33 0.33 0.33 0.32 0.32

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Use of Peptide Analogue Diversity Library Beads Table 2. Continued protein

Mr

Uniprot ID

TSC

% abun.

apolipoprotein H amyloid precursor-like protein 1 isoform 2 * complement component 4 binding protein insulin-like growth factor binding protein 2, 36 kDa haptoglobin kininogen 1 VGF nerve growth factor inducible complement component 9 fibrinogen, gamma chain isoform gamma-B fibronectin 1 isoform 3 preproprotein PREDICTED: similar to Ig gamma-1 chain C region alpha-2-HS-glycoprotein complement component 1, s subcomponent * complement component 1, q subcomponent, gamma * Crystallin, alpha B * complement component 1, q subcomponent, A chain PREDICTED: similar to Ig gamma-4 chain C region neuronal cell adhesion molecule isoform B * phospholipid transfer protein isoform A * nel-like 2 * ATPase, H+ transporting, lysosomal accessory protein * secretogranin II neural cell adhesion molecule 1 isoform 1 * procollagen C-endopeptidase enhancer secreted phosphoprotein 1 isoform A retinol-binding protein 4 fibrinogen, beta chain preproprotein interalpha (globulin) inhibitor H4 complement component 7 * Crystallin, beta B2 * ribonuclease, RNase A family, 4 vitronectin autotaxin isoform 2 preproprotein EGF-containing fibulin-like extracellular matrix beta-2-microglobulin * prostatic binding protein * cell growth regulator with EF hand domain 1 * Crystallin, beta A3 PREDICTED: similar to Ig gamma-3 chain C region * apolipoprotein C-II cell adhesion molecule with homology to L1CAM * plasma glutathione peroxidase 3 * carboxypeptidase E lysozyme UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltra chitotriosidase alpha-1-microglobulin/bikunin * amyloid beta A4 protein, isoform A calsyntenin 1 isoform 1 pancreatic ribonuclease chromogranin B interalpha globulin inhibitor H2 polypeptide fibulin 1 isoform B * complement factor H-related 1 * heparin cofactor II * lumican alpha-2-glycoprotein 1, zinc * proenkephalin * GM2 ganglioside activator * nter-alpha (globulin) inhibitor H1 * dermcidin preproprotein PREDICTED: similar to Ig gamma-2 chain C region fibulin 1 isoform D * seizure related 6 homologue * PREDICTED: similar to Fc fragment of IgG binding protein * transforming growth factor, beta-induced, 68 kDa

38286.68 72131.33 66989.44 35114.41 45176.59 47852.68 67217.77 63132.78 51478.88 259061 64280.8 39299.73 76634.85 25757.13 20146.43 26000.19 47414.69 130964.4 54704.67 91284.31 51992.65 70897.13 93302.63 47915.98 35401.25 22995.26 55892.23 103293.2 93457.27 23365.42 16829.37 54271.23 98929.59 54604.29 13705.91 21043.67 31886.1 25133.82 18069.91 11276.75 136612.7 35385.95 53117.17 16526.29 47088.94 51648.38 38973.99 86888.16 109723.7 17632.68 78199.14 106396.8 65427.26 37636.97 57034.27 38404.8 34237.13 30767.02 20824.73 101338.8 11276.83 39968.81 77190.46 107441.2 30792.19 74634.1

P02749 Q5T012 Q5VVQ8 P18065 P00738 P01042 Q9UDW8 Q9UDW8 068656 P02751 652050 P02765 Q53HU9 Q5T960 Q9UJY1 Q5T960 P01861 A4D0S3 Q53H91 Q99435 Q4R500 P13521 P13592 Q15113 000571 P02753 P02675 Q59FS1 Q8TCS7 P02511 P61221 P04004 1035181 Q12805 Q9UM88 P30086 P30086 P05813 947052 P02655 Q59FY0 P22352 P16870 Q8N1E2 O43505 Q13231 Q5TBD7 P05067 O94985 P07998 P05060 A2RTY6 Q59G97 Q5TMF4 P05546 P51884 P02765 P01213 P17900 Q59FS1 P81605 P01859 Q96K89 Q53EL9 P01876 Q15582

17 32 29 15 19 20 28 26 21 96 23 14 27 9 7 9 16 44 18 30 17 23 30 15 11 7 17 31 28 7 5 16 29 16 4 6 9 7 5 3 35 9 13 4 11 12 9 20 25 4 17 23 14 8 12 8 7 6 4 19 2 7 13 18 5 12

0.32 0.32 0.31 0.31 0.30 0.30 0.30 0.30 0.29 0.27 0.26 0.26 0.25 0.25 0.25 0.25 0.24 0.24 0.24 0.24 0.23 0.23 0.23 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.21 0.21 0.21 0.21 0.21 0.20 0.20 0.20 0.20 0.19 0.18 0.18 0.18 0.17 0.17 0.17 0.17 0.17 0.16 0.16 0.16 0.16 0.15 0.15 0.15 0.15 0.15 0.14 0.14 0.13 0.13 0.13 0.12 0.12 0.12 0.12

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research articles

Shores et al.

Table 2. Continued protein

Mr

Uniprot ID

TSC

% abun.

* fibrinogen, alpha polypeptide isoform A * leucine-rich alpha-2-glycoprotein 1 * PREDICTED: similar to Ceruloplasmin * complement component 5 * alpha 1 type I collagen preproprotein * complement component 2 * neogenin homologue 1 * lactate dehydrogenase B prion protein preproprotein * Crystallin, beta B1 * hypothetical protein LOC146556 * interalpha (globulin) inhibitor H3 * cartilage acidic protein 1 * complement component 8, beta polypeptide * Crystallin, beta A4 C-type lectin domain family 3, member B * contactin 1 isoform 1 * gap junction protein, alpha 8, 50 kDa * Crystallin, beta B3 * PREDICTED: similar to Pyruvate kinase, isozyme * apolipoprotein B * coagulation factor XII precursor complement component 4B preproprotein * dystroglycan 1 * alpha 2 type IV collagen preproprotein Fc fragment of IgG binding protein * microfibrillar-associated protein 4 * Complement component 6 * PREDICTED: similar to Carboxypeptidase N subunit 2 * alpha 2 type I collagen brevican isoform 1 * nudix -type motif 12 * matrix metalloproteinase 2 preproprotein * aldolase A * fructose-bisphosphate aldolase C * Actin, gamma 1 propeptide * ADAM metallopeptidase with thrombospondin type 1 * collagen, type VI, alpha 1 * apolipoprotein L1 isoform A * golgi phosphoprotein 2 * immunoglobulin superfamily containing leucine-rich afamin * plasma kallikrein B1 precursor * calcium channel, voltage-dependent, alpha 2/delta * alpha 1 type XVIII collagen isoform 3 * protein S (alpha) * lipopolysaccharide-binding protein * vimentin * contactin 2 * I factor (complement) * insulin-like growth factor binding protein, acid labile subunit insulin-like growth factor binding protein 7 * transmembrane protein 132A isoform A fibulin 1 isoform C * RAN binding protein 5 gelsolin isoform A * laminin, gamma 1 * nidogen (enactin) * karyopherin beta 1 * peptidylglycine alpha-amidating monooxygenase * heparan sulfate proteoglycan 2 * exportin 6 * mannosidase, alpha, class 2A, member 2 * nidogen 2 * alpha 3 type VI collagen isoform 2 * tenascin XB isoform 1 * coagulation factor V * reelin isoform B

69713.77 38154.13 32078.9 188185.3 138826.9 83214.38 159859.3 36615.16 27643.19 28005.89 48734.75 99786.66 71375.93 66904.45 22359.61 22552.29 113249.1 48198.64 24236.94 39439.27 515209.6 67774.05 192629.6 97519.91 167361.3 571719.8 28629.89 104775.9 60546.27 129235.8 99056.16 52042.77 73834.79 39395.32 39431.26 41765.8 105289.9 108462 43946.95 45305.82 45968.42 69024.09 71295.82 123105.6 149840 75024.04 53350.02 53619.17 113393.8 65724.66 65993.97 65993.97 110128.6 74412.02 125463.8 85644.25 177457.8 136402.7 97108.17 100754.1 468528.2 128800.9 130456.8 151199 325090.4 464034.9 251543.8 387950.7

P02671 P02750 877964 Q27I61 Q6LAN8 A2AAQ4 Q59FP8 P07195 P04156 P53674 Q8N213 Q53F06 Q9NQ79 A1L4K7 P53673 P05452 Q12860 Q5VVN9 P26998 652797 P41238 P00488 Q6U2E9 Q14118 Q9Y5P4 P01876 Q4W5N7 P13671 P22792 Q9Y5P4 Q59F90 Q9BQG2 P08253 P04075 P09972 P63261 A1L3U9 Q8N4Z1 O14791 Q9H8Y8 O14498 P43652 Q4W5C3 Q17R45 Q2UY07 P31946 P18428 P08670 Q02246 Q4R955 Q8TAY0 Q16270 Q24JP5 Q8N9G0 Q13451 A2A418 P11047 P14543 Q14974 620121 Q5SZI5 Q96QU8 Q16706 Q14112 Q8N4Z1 Q59GU7 P12259 P78509

11 6 5 26 17 10 18 4 3 3 5 10 7 6 2 2 10 4 2 3 39 5 14 7 12 40 2 7 4 8 6 3 4 2 2 2 5 5 2 2 2 3 3 5 6 3 2 2 4 2 2 2 3 2 3 2 4 3 2 2 9 2 2 2 4 4 2 2

0.11 0.11 0.11 0.10 0.09 0.09 0.08 0.08 0.08 0.08 0.07 0.07 0.07 0.06 0.06 0.06 0.06 0.06 0.06 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.04 0.04 0.04 0.04 0.04 0.04 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.00

a Relative abundance was calculated by normalizing the total spectrum count (TSC) of a protein by its molecular weight (Mr). Proteins identified only in the depleted CSF sample are indicated by asterisks.

and 85), and a majority of proteins that were uniquely identified in the depleted sample (indicated by asterisks in Table 2) are 1930

Journal of Proteome Research • Vol. 7, No. 5, 2008

of low relative abundance. Furthermore, approximately 95% of the all unique proteins identified in the control and depleted

research articles

Use of Peptide Analogue Diversity Library Beads

Shores was supported by NIH T32 HL007260. We thank Dr. J. Baatz for protein assays.

Figure 4. Proteins identified in the four shotgun analyses of the peptoid depleted CSF and in three shotgun analyses of the control CSF sample.

sample (210) were identified in the depleted sample (Figure 4), suggesting that very few proteins may have failed to bind to the peptoid library. Moreover, since depletion with the peptoid library is a “bind and elute” method, contaminants that are less prone to bind to the peptoid beads than the proteins in the sample may be removed during the washing process. In this manner, depletion with the peptoid library may serve to purify the sample which could also contribute to a greater number of protein identifications. The theoretical number of different ligands present in the hexapeptoid library is approximately 262 000. Sennels et al. recently used a hexapeptide library with far greater diversity (few dozen of millions of different ligands) to deplete human serum and identified more than 1559 unique proteins.17 The diversity of the peptide library required Sennels et al. to process 300 µL of serum (the protein concentration of human serum is approximately 200 times greater than that of human CSF3), since the saturation limit for an individual protein in the hexapeptide library is far lower than that of the hexapeptoid library used in this work. If we had used a hexapeptoid library with greater diversity, we predict that a greater number of lowabundance proteins would have been identified; however, due to the dilute nature of human CSF, a much larger sample size would have been required. Nonetheless, depletion with the hexapeptoid library has led to a significantly greater number of protein IDs and is an encouraging step toward development of a method of high abundance CSF protein depletion.

4. Conclusions This work demonstrates a method of depleting highabundance proteins from CSF by reducing the overall dynamic range of concentrations using peptoid library beads. Following application of the depletion method to a pooled CSF sample and 2D LC/MS/MS analysis, 191 unique proteins were identified after three consecutive analyses, while only 115 proteins were identified after three analyses of the undepleted sample. A total of 200 unique proteins were identified in four consecutive analyses of the depleted CSF sample. The reported method thus enables observation of more proteins in this relatively dilute biological fluid than is possible in the untreated sample. The use of the peptoid bead diversity library thus gives improvement in proteomic analysis analogous to that seen with the previously reported method using peptide diversity library beads,1 but with the expected advantages of resistance of the beads to degradation by proteases in the samples or by bacterial action on storage, as well as simpler and less expensive preparation of the beads. Peptoid diversity library beads could thus offer a useful tool for improved proteomic analysis.

Acknowledgment. This work was supported in part by the NHLBI Proteomics Initiative via N01-HV-28181. Kevin S.

Supporting Information Available: Tables with a complete list of the proteins identified in the control CSF sample and the peptoid depleted CSF sample. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Righetti, P. G.; Boschetti, E.; Lomas, L.; Citterio, A. Protein Equalizer Technology: the quest for a “democratic proteome”. Proteomics 2006, 6 (14), 3980–3992. (2) Anderson, N. L.; Anderson, N. G. The human plasma proteome: history, character, and diagnostic prospects. Mol. Cell. Proteomics 2002, 1 (11), 845–867. (3) Huhmer, A. F.; Biringer, R. G.; Amato, H.; Fonteh, A. N.; Harrington, M. G. Protein analysis in human cerebrospinal fluid: Physiological aspects, current progress and future challenges. Dis. Markers 2006, 22 (1–2), 3–26. (4) Gianazza, E.; Arnaud, P. Chromatography of plasma proteins on immobilized Cibacron Blue F3-GA. Mechanism of the molecular interaction. Biochem. J. 1982, 203 (3), 637–641. (5) 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 (2), 125-133; discussion 173–80. (6) Noben, J. P.; Dumont, D.; Kwasnikowska, N.; Verhaert, P.; Somers, V.; Hupperts, R.; Stinissen, P.; Robben, J. Lumbar cerebrospinal fluid proteome in multiple sclerosis: characterization by ultrafiltration, liquid chromatography, and mass spectrometry. J. Proteome Res. 2006, 5 (7), 1647–157. (7) 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 (14), 2402– 2412. (8) 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), 837–845. (9) 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), 3739–3751. (10) Guerrier, L.; Claverol, S.; Fortis, F.; Rinalducci, S.; Timperio, A. M.; Antonioli, P.; Jandrot-Perrus, M.; Boschetti, E.; Righetti, P. G. Exploring the platelet proteome via combinatorial, hexapeptide ligand libraries. J. Proteome Res. 2007, 6 (11), 4290–4303. (11) Simon, R. J.; Kania, R. S.; Zuckermann, R. N.; Huebner, V. D.; Jewell, D. A.; Banville, S.; Ng, S.; Wang, L.; Rosenberg, S.; Marlowe, C. K.; et al. Peptoids: a modular approach to drug discovery. Proc. Natl. Acad. Sci. U.S.A. 1992, 89 (20), 9367–9371. (12) Figliozzi, G. M.; Goldsmith, R.; Ng, S. C.; Banville, S. C.; Zuckermann, R. N. Synthesis of N-substituted glycine peptoid libraries. Methods Enzymol. 1996, 267, 437–447. (13) Kodadek, T.; Reddy, M. M.; Olivos, H. J.; Bachhawat-Sikder, K.; Alluri, P. G. Synthetic molecules as antibody replacements. Acc. Chem. Res. 2004, 37 (9), 711–8. (14) Eng, J. K.; McCormack, A. L.; Yates, J. R., III. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 1994, 5 (11), 976–989. (15) Liu, H.; Sadygov, R. G.; Yates, J. R., III. A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal. Chem. 2004, 76 (14), 4193–4201. (16) Kislinger, T.; Gramolini, A. O.; MacLennan, D. H.; Emili, A. Multidimensional protein identification technology (MudPIT): technical overview of a profiling method optimized for the comprehensive proteomic investigation of normal and diseased heart tissue. J. Am. Soc. Mass Spectrom. 2005, 16 (8), 1207–1220. (17) Sennels, L.; Salek, M.; Lomas, L.; Boschetti, E.; Righetti, P. G.; Rappsilber, J. Proteomic analysis of human blood serum using peptide library beads. J. Proteome Res. 2007, 6 (10), 4055–4062.

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