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18O-Labeled Proteome Reference as Global Internal Standards for Targeted Quantification by Selected Reaction Monitoring-Mass Spectrometry

2011; Elsevier BV; Volume: 10; Issue: 12 Linguagem: Inglês

10.1074/mcp.m110.007302

1535-9484

Jong‐Seo Kim, Thomas Fillmore, Tao Liu, Errol Robinson, Mahmud Hossain, Boyd L. Champion, Ronald J. Moore, David Camp, Richard Smith, Weijun Qian,

Metabolomics and Mass Spectrometry Studies

Selected reaction monitoring (SRM)-MS is an emerging technology for high throughput targeted protein quantification and verification in biomarker discovery studies; however, the cost associated with the application of stable isotope-labeled synthetic peptides as internal standards can be prohibitive for screening a large number of candidate proteins as often required in the preverification phase of discovery studies. Herein we present a proof of concept study using an 18O-labeled proteome reference as global internal standards (GIS) for SRM-based relative quantification. The 18O-labeled proteome reference (or GIS) can be readily prepared and contains a heavy isotope (18O)-labeled internal standard for every possible tryptic peptide. Our results showed that the percentage of heavy isotope (18O) incorporation applying an improved protocol was >99.5% for most peptides investigated. The accuracy, reproducibility, and linear dynamic range of quantification were further assessed based on known ratios of standard proteins spiked into the labeled mouse plasma reference. Reliable quantification was observed with high reproducibility (i.e. coefficient of variance 99.5% for most peptides investigated. The accuracy, reproducibility, and linear dynamic range of quantification were further assessed based on known ratios of standard proteins spiked into the labeled mouse plasma reference. Reliable quantification was observed with high reproducibility (i.e. coefficient of variance <10%) for analyte concentrations that were set at 100-fold higher or lower than those of the GIS based on the light (16O)/heavy (18O) peak area ratios. The utility of 18O-labeled GIS was further illustrated by accurate relative quantification of 45 major human plasma proteins. Moreover, quantification of the concentrations of C-reactive protein and prostate-specific antigen was illustrated by coupling the GIS with standard additions of purified protein standards. Collectively, our results demonstrated that the use of 18O-labeled proteome reference as GIS provides a convenient, low cost, and effective strategy for relative quantification of a large number of candidate proteins in biological or clinical samples using SRM. Selected reaction monitoring (SRM), 1The abbreviations used are:SRMselected reaction monitoringGISglobal internal standardsPSAprostate-specific antigenCRPC-reactive protein. 1The abbreviations used are:SRMselected reaction monitoringGISglobal internal standardsPSAprostate-specific antigenCRPC-reactive protein. also known as multiple reaction monitoring, is a promising technology for reliable quantification of targeted analytes in complex biological and clinical applications (1Anderson L. Hunter C.L. 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The precision and reproducibility of SRM-based protein quantification in human plasma applying labeled synthetic peptides as internal standards have been recently demonstrated through a multi-site assessment (11Addona T.A. Abbatiello S.E. Schilling B. Skates S.J. Mani D.R. Bunk D.M. Spiegelman C.H. Zimmerman L.J. Ham A.J. Keshishian H. Hall S.C. Allen S. Blackman R.K. Borchers C.H. Buck C. Cardasis H.L. Cusack M.P. Dodder N.G. Gibson B.W. Held J.M. Hiltke T. Jackson A. Johansen E.B. Kinsinger C.R. Li J. Mesri M. Neubert T.A. Niles R.K. Pulsipher T.C. Ransohoff D. Rodriguez H. Rudnick P.A. Smith D. Tabb D.L. Tegeler T.J. Variyath A.M. Vega-Montoto L.J. Wahlander A. Waldemarson S. Wang M. Whiteaker J.R. Zhao L. Anderson N.L. Fisher S.J. Liebler D.C. Paulovich A.G. Regnier F.E. Tempst P. Carr S.A. Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma.Nat. Biotechnol. 2009; 27: 633-641Crossref PubMed Scopus (862) Google Scholar). However, the technology is presently limited in the number of candidate proteins that can be screened often because of the costs associated with stable isotope-labeled synthetic peptides (1Anderson L. Hunter C.L. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins.Mol. Cell. Proteomics. 2006; 5: 573-588Abstract Full Text Full Text PDF PubMed Scopus (1078) Google Scholar, 3Keshishian H. Addona T. Burgess M. Kuhn E. Carr S.A. Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution.Mol. Cell. Proteomics. 2007; 6: 2212-2229Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar, 7Picotti P. Bodenmiller B. Mueller L.N. Domon B. Aebersold R. Full dynamic range proteome analysis of S. cerevisiae by targeted proteomics.Cell. 2009; 138: 795-806Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar, 12Wu C.C. MacCoss M.J. Howell K.E. Matthews D.E. Yates 3rd, J.R. Metabolic labeling of mammalian organisms with stable isotopes for quantitative proteomic analysis.Anal. Chem. 2004; 76: 4951-4959Crossref PubMed Scopus (339) Google Scholar, 13Ishihama Y. Sato T. Tabata T. Miyamoto N. Sagane K. Nagasu T. Oda Y. Quantitative mouse brain proteomics using culture-derived isotope tags as internal standards.Nat. Biotechnol. 2005; 23: 617-621Crossref PubMed Scopus (195) Google Scholar) or artificial protein concatemers (1Anderson L. Hunter C.L. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins.Mol. Cell. Proteomics. 2006; 5: 573-588Abstract Full Text Full Text PDF PubMed Scopus (1078) Google Scholar, 14Beynon R.J. Doherty M.K. Pratt J.M. Gaskell S.J. Multiplexed absolute quantification in proteomics using artificial QCAT proteins of concatenated signature peptides.Nat. Methods. 2005; 2: 587-589Crossref PubMed Scopus (393) Google Scholar, 15Rivers J. Simpson D.M. Robertson D.H. Gaskell S.J. Beynon R.J. Absolute multiplexed quantitative analysis of protein expression during muscle development using QconCAT.Mol. Cell. Proteomics. 2007; 6: 1416-1427Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) that are typically utilized as internal standards. Recently the use of 18O labeling for individual synthetic peptides as internal standards for SRM has also been recently reported (16Zhao Y. Jia W. Sun W. Jin W. Guo L. Wei J. Ying W. Zhang Y. Xie Y. Jiang Y. He F. Qian X. Combination of improved O-18 incorporation and multiple reaction monitoring: A universal strategy for absolute quantitative verification of serum candidate biomarkers of liver cancer.J. Proteome Res. 2010; 9: 3319-3327Crossref PubMed Scopus (50) Google Scholar). In this work, we investigated the concept of using an 18O-labeled proteome reference to generate global internal standards (GIS) as an alternative to stable isotope-labeled synthetic peptides for broad SRM-based relative quantification. selected reaction monitoring global internal standards prostate-specific antigen C-reactive protein. selected reaction monitoring global internal standards prostate-specific antigen C-reactive protein. The initial concept of employing internal standards derived from a stable isotope-labeled whole proteome has been demonstrated previously for global quantitative proteomics (17Petyuk V.A. Qian W.J. Chin M.H. Wang H. Livesay E.A. Monroe M.E. Adkins J.N. Jaitly N. Anderson D.J. Camp 2nd, D.G. Smith D.J. Smith R.D. Spatial mapping of protein abundances in the mouse brain by voxelation integrated with high-throughput liquid chromatography-mass spectrometry.Genome Res. 2007; 17: 328-336Crossref PubMed Scopus (55) Google Scholar, 18Qian W.J. Liu T. Petyuk V.A. Gritsenko M.A. Petritis B.O. Polpitiya A.D. Kaushal A. Xiao W. Finnerty C.C. Jeschke M.G. Jaitly N. Monroe M.E. Moore R.J. Moldawer L.L. Davis R.W. Tompkins R.G. Herndon D.N. Camp D.G. Smith R.D. Large-scale multiplexed quantitative discovery proteomics enabled by the use of an (18)O-labeled "universal" reference sample.J. Proteome Res. 2009; 8: 290-299Crossref PubMed Scopus (50) Google Scholar, 19Qian W.J. Petritis B.O. Kaushal A. Finnerty C.C. Jeschke M.G. Monroe M.E. Moore R.J. Schepmoes A.A. Xiao W. Moldawer L.L. Davis R.W. Tompkins R.G. Herndon D.N. Camp 2nd, D.G. Smith R.D. Plasma proteome response to severe burn injury revealed by O-18-labeled "universal" reference-based quantitative proteomics.J. Proteome Res. 2010; 9: 4779-4789Crossref PubMed Scopus (47) Google Scholar). In one case, a "universal" reference was created by pooling aliquots of tryptically digested individual samples, and then the peptides in the pooled sample were labeled with 18O. 18O labeling is ideal for generating a labeled reference sample because of its simplicity, versatility in labeling any kind of biological sample, and low cost. When spiked into individual samples, the 18O-labeled proteome reference served as GIS for all tryptic peptides that enabled relative abundance comparisons across any number of samples based on light/heavy (16O/18O) ratios (18Qian W.J. Liu T. Petyuk V.A. Gritsenko M.A. Petritis B.O. Polpitiya A.D. Kaushal A. Xiao W. Finnerty C.C. Jeschke M.G. Jaitly N. Monroe M.E. Moore R.J. Moldawer L.L. Davis R.W. Tompkins R.G. Herndon D.N. Camp D.G. Smith R.D. Large-scale multiplexed quantitative discovery proteomics enabled by the use of an (18)O-labeled "universal" reference sample.J. Proteome Res. 2009; 8: 290-299Crossref PubMed Scopus (50) Google Scholar). In the present study, known ratios of standard proteins spiked into mouse plasma were used to evaluate the accuracy, reproducibility, and dynamic range of SRM-based quantification using the 18O-labeled GIS. The benefits of the labeled proteome reference were further illustrated by accurate relative quantification of 45 major human plasma proteins without the use of synthetic peptides (1Anderson L. Hunter C.L. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins.Mol. Cell. Proteomics. 2006; 5: 573-588Abstract Full Text Full Text PDF PubMed Scopus (1078) Google Scholar, 6Kuzyk M.A. Smith D. Yang J. Cross T.J. Jackson A.M. Hardie D.B. Anderson N.L. Borchers C.H. Multiple reaction monitoring-based, multiplexed, absolute quantitation of 45 proteins in human plasma.Mol. Cell. Proteomics. 2009; 8: 1860-1877Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). Quantification of endogenous C-reactive protein (CRP) and spiked prostate-specific antigen (PSA) concentrations in nondepleted human female plasma was achieved by using a standard addition method with purified protein standards and the 18O-labeled reference. Our results demonstrate that the 18O-labeled proteome reference affords sufficient quantification accuracy for SRM analysis of many protein targets in biological samples. Mouse plasma (∼40 mg/ml determined by BCA protein assay (Pierce)) obtained from Equitech-Bio, Inc. (Kerrville, TX), human female plasma (∼50 mg/ml) obtained from BioChemed Services (Winchester, VA), six nonhuman standard proteins obtained from Sigma-Aldrich, i.e. bovine carbonic anhydrase II, bovine β-lactoglobulin, Escherichia coli β-galactosidase, equine skeletal muscle myoglobin, chicken ovalbumin, and bovine cytochrome c, and two human standard proteins PSA obtained from Sigma, and CRP obtained from EMD Chemicals (Gibbstown, NJ) were used in this study. In the experiment for quantifying "endogenous" protein concentrations, three human female plasma samples were prepared with PSA and CRP at three different concentrations (2, 4, and 10 μg/ml), respectively; another human female plasma sample spiked with 0.50 μg/ml PSA only was used as a "hypothetical" endogenous sample. All of the protein samples including the nonhuman standard proteins, mouse plasma, and human plasma were digested individually. The protein samples were initially denatured and reduced with 8 m urea and 10 mm dithiothreitol in 50 mm NH4HCO3 buffer (pH 8.2) for 1 h at 37 °C and followed by alkylation of cysteine residues with 40 mm iodoacetamide for 1 h at 37 °C in the dark. Following a 10-fold dilution with 50 mm NH4HCO3, each resulting sample was digested separately using sequencing grade modified porcine trypsin (Promega, Madison, WI) at a trypsin-to-protein ratio of 1:50 (w/w) for 5 h at 37 °C. The digested samples were loaded individually onto a 1-ml solid phase extraction C18 column (Supelco, Bellefonte, PA) and washed with 4 ml of 0.1% trifluoroacetic acid, 5% acetonitrile. Peptides were eluted from the solid phase extraction column with 1 ml of 0.1% trifluoroacetic acid, 80% acetonitrile and then lyophilized. After reconstituting the resulting peptide samples in 25 mm NH4HCO3, the residual trypsin activity was quenched by boiling for 10 min, and then the samples were immediately placed on ice for 30 min. The final peptide concentration for each sample was measured with the BCA protein assay. Two 18O-labeled mouse plasma reference samples were generated by spiking six standard protein digests into mouse plasma digests at 0.1% and 0.01% (w/w) of the total mouse plasma protein mass. The corresponding standard protein concentrations in original plasma were ∼40 and ∼4 μg/ml, respectively. Trypsin-catalyzed 18O labeling at the peptide level was performed using a recently improved protocol (20Petritis B.O. Qian W.J. Camp 2nd, D.G. Smith R.D. A simple procedure for effective quenching of trypsin activity and prevention of 18O-labeling back-exchange.J. Proteome Res. 2009; 8: 2157-2163Crossref PubMed Scopus (48) Google Scholar). Briefly, the peptide sample was lyophilized to dryness and reconstituted in 100 μl of 50 mm NH4HCO3 in H218O (97%; ISOTEC, Miamisburg, OH), pH 7.8. One μl of 1 m CaCl2 and solution phase trypsin dissolved in H218O at a 1:50 trypsin/peptide ratio (w/w) were added to the samples. The tubes were wrapped in parafilm and mixed continuously for 5 h at 37 °C. The reaction was stopped by boiling the sample in a water bath for 10 min. After snap-freezing the sample in liquid nitrogen, the samples were acidified by adding 5 μl of formic acid, and final peptide concentrations were measured using a BCA assay. Similarly, a human female plasma sample spiked with PSA (2 μg/ml) and CRP (2 μg/ml) was digested and labeled as the 18O-reference. In the experiments for quantifying 45 human plasma proteins, a labeled human plasma digest was prepared with 99% 18O-enriched water (Cambridge Isotope Laboratories, Andover, MA) rather than the initial stock of 97% 18O-enriched water. Individually digested standard proteins were spiked into the two labeled mouse plasma references to generate 11 calibration mixtures from 400 ng/ml to 400 μg/ml for the unlabeled spiked proteins in the labeled plasma references with the unlabeled versus labeled protein concentration ratios ranging from 0.01 to 100 (see Table I). For quantifying 45 major human plasma proteins, a labeled human plasma reference and unlabeled plasma digest were prepared separately. The unlabeled human plasma digest and the labeled reference were then mixed 1:10, 1:3, 1:1, 3:1, and 10:1 peptide mass ratios of the unlabeled versus the labeled reference to make five mixtures. All of the samples were analyzed by LC-SRM-MS in three technical replicates (triplicate injections).Table IA series of calibration mixtures made of mouse plasma and six standard proteins. The concentration values denote the concentrations in original plasmaCalibration mixtureConcentration of each standard protein in 18O-reference (% w/w)Concentration of each unlabeled standard proteinStandard protein concentration ratio (unlabeled: 18O-reference)M040 μg/ml (0.10%)BlankBackgroundM140 μg/ml (0.10%)400 ng/ml1:100M240 μg/ml (0.10%)800 ng/ml1:50M340 μg/ml (0.10%)1.6 μg/ml1:25M440 μg/ml (0.10%)4 μg/ml1:10M540 μg/ml (0.10%)8 μg/ml1:5M640 μg/ml (0.10%)40 μg/ml1:1M74 μg/ml (0.010%)20 μg/ml5:1M84 μg/ml (0.010%)40 μg/ml10:1M94 μg/ml (0.010%)100 μg/ml25:1M104 μg/ml (0.010%)200 μg/ml50:1M114 μg/ml (0.010%)400 μg/ml100:1 Open table in a new tab A total of 79 peptides from the six nonhuman standard proteins were preselected by in silico screening. The minimum length and maximum molecular weight of target tryptic peptide sequences were eight amino acid residues and 2400 Da, respectively. Most target peptides are without any missed cleavages with the exception of a few high responding peptides that contain partial sequences of KD or KK, which were shown to significantly inhibit trypsin activity (21Riviere L.R. Tempst P. Enzymatic digestion of proteins in solution.Curr. Protoc. Protein Sci. 2001; : 11.1.1-11.1.19Google Scholar). Similarly, 16 peptides for PSA/CRP (eight peptides for each protein) were preselected. Initially, transitions of each selected peptide and its optimal collision energy were obtained from infusion experiments in which 500 fmol/μl solution of each standard protein digest in a 1:1 mixture of water and acetonitrile containing 0.1% formic acid were infused at a flow rate of 300 nl/min. The three most intense transitions of y-type ions were utilized for SRM quantification for each peptide. All of the peptides spiked in the mouse and human plasma digests were screened and validated by LC-SRM-MS, and at least two of the most responsive peptides per protein in the SRM mode were selected for the assessment of quantification. Moreover, six peptides, i.e. bradykinin fragments 1–7, kemptide, melittin, methionine enkephalin, renin substrate porcine, and [d-Ala2]-deltorphin II (Sigma), were used as quality control peptides (two optimized transitions per peptide) for monitoring the overall performance of the platform and for LC retention time markers. For quality control peptides without tryptic terminal, both b- and y-type transitions were considered. All of the preselected peptides and parameters of SRM screened peptides are summarized in supplemental Table 1. For the 45 major human plasma proteins monitored by Kuzyk et al. (6Kuzyk M.A. Smith D. Yang J. Cross T.J. Jackson A.M. Hardie D.B. Anderson N.L. Borchers C.H. Multiple reaction monitoring-based, multiplexed, absolute quantitation of 45 proteins in human plasma.Mol. Cell. Proteomics. 2009; 8: 1860-1877Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar), we selected 24 of the original 45 peptides without C-terminal inhibitory motifs. The other 21 peptides were found to contain inhibitory motifs (e.g. RK, KK, (D/E)K, or K(D/E)) for trypsin activity at their C termini (21Riviere L.R. Tempst P. Enzymatic digestion of proteins in solution.Curr. Protoc. Protein Sci. 2001; : 11.1.1-11.1.19Google Scholar, 22Yen C.Y. Russell S. Mendoza A.M. Meyer-Arendt K. Sun S. Cios K.J. Ahn N.G. Resing K.A. Improving sensitivity in shotgun proteomics using a peptide-centric database with reduced complexity: Protease cleavage and SCX elution rules from data mining of MS/MS spectra.Anal. Chem. 2006; 78: 1071-1084Crossref PubMed Scopus (67) Google Scholar, 23Siepen J.A. Keevil E.J. Knight D. Hubbard S.J. Prediction of missed cleavage sites in tryptic peptides aids protein identification in proteomics.J. Proteome Res. 2007; 6: 399-408Crossref PubMed Scopus (121) Google Scholar), which could lead to ineffective 18O labeling. Therefore, these peptides were replaced by other peptides from the same proteins. A total of 42 peptides without such motifs for these 21 proteins were initially selected using the Skyline software tool (24MacLean B. Tomazela D.M. Shulman N. Chambers M. Finney G.L. Frewen B. Kern R. Tabb D.L. Liebler D.C. MacCoss M.J. Skyline: An open source document editor for creating and analyzing targeted proteomics experiments.Bioinformatics. 2010; 26: 966-968Crossref PubMed Scopus (2964) Google Scholar) with a spectrum library based on previous LC-MS/MS data. All of the transitions were selected based on ion trap MS/MS spectra without further optimization because it has been reported the intensity order of transition is well correlated between ion trap CID and SRM (25Sherwood C.A. Eastham A. Lee L.W. Risler J. Vitek O. Martin D.B. Correlation between y-type ions observed in ion trap and triple quadrupole mass spectrometers.J. Proteome Res. 2009; 8: 4243-4251Crossref PubMed Scopus (46) Google Scholar). The predicted collision energies from Skyline were used for all peptides. After LC-SRM-MS screening, a total of 45 peptides for the 45 major plasma proteins (one peptide per protein) were selected for relative quantification in this study (see Table II).Table IISRM quantification of 45 human plasma proteins with known relative abundance ratios relative to the labeled referenceProteinPeptidePeptide replacedProduct ionSlope ± CIaCI, 95% confidence interval.y intercept ± CIaCI, 95% confidence interval.R2Accuracy (Error %)bAccuracy was calculated based on the percentage of error relative to the known relative abundance ratios.Mean % CVAfaminDADPDTFFAKy7+1.013 ± 0.0180.036 ± 0.0860.9993.453.41Albumin, serumLVNEVTEFAKy5+0.968 ± 0.0320.053 ± 0.1510.9972.172.42α1-Acid glycoprotein 1EQLGEFYEALDCLR√y8+0.870 ± 0.0280.137 ± 0.1300.9976.822.39α1-AntichymotrypsinEIGELYLPKy2+1.023 ± 0.0070.025 ± 0.0321.0002.411.62α1B-GlycoproteinLETPDFQLFKy8+0.948 ± 0.0280.141 ± 0.1310.9986.602.66α2-AntiplasminLCQDLGPGAFR√y6+0.984 ± 0.0290.091 ± 0.1350.9984.043.27α2-MacroglobulinLLIYAVLPTGDVIGDSAKy11+0.985 ± 0.0270.110 ± 0.1290.9988.362.04AngiotensinogenALQDQLVLVAAKy3+0.982 ± 0.0210.068 ± 0.0970.9994.663.52Antithrombin-IIIDDLYVSDAFHKy6+0.989 ± 0.0540.040 ± 0.2510.9925.486.06Apolipoprotein A-ILLDNWDSVTSTFSK√y8+0.976 ± 0.0120.058 ± 0.0551.0004.641.06Apolipoprotein A-II precursorSPELQAEAKy8+0.947 ± 0.0280.094 ± 0.1310.9986.163.43Apolipoprotein A-IVALVQQMEQLR√y6+1.003 ± 0.0130.033 ± 0.0591.0002.542.70Apolipoprotein B-100LTISEQNIQR√y7+0.993 ± 0.0170.056 ± 0.0780.9994.034.76Apolipoprotein C-I lipoproteinEWFSETFQK√y7+1.090 ± 0.0370.060 ± 0.1740.9973.244.75Apolipoprotein C-IIIDALSSVQESQVAQQAR√y10+1.025 ± 0.0170.088 ± 0.0810.9995.362.97Apolipoprotein ELGPLVEQGRy7+1.027 ± 0.0220.017 ± 0.1020.9993.022.73β2-Glycoprotein IATVVYQGERy6+0.999 ± 0.0060.032 ± 0.0291.0003.331.43CeruloplasminEVGPTNADPVCLAK√y6+0.997 ± 0.0260.037 ± 0.1240.9982.971.68ClusterinELDESLQVAERy3+0.978 ± 0.0220.050 ± 0.1040.9994.763.22Coagulation factor XIIa HCVVGGLVALRy7+0.969 ± 0.0170.066 ± 0.0810.9996.171.93Complement C3AVLYNYR√y5+0.987 ± 0.0080.03 ± 0.0391.0002.691.32Complement C4 β chainVGDTLNLNLRy3+0.986 ± 0.0090.022 ± 0.0401.0002.452.1Complement C4 γ chainVEYGFQVK√y6+1.024 ± 0.0150.001 ± 0.0711.0003.692.02Complement component C9VVEESELAR√y7+1.014 ± 0.0230.075 ± 0.1080.9995.492.61Complement factor BLEDSVTYHCSR√y6+0.984 ± 0.0480.002 ± 0.2230.9943.443.94Complement factor HEIMENYNIALR√y7+0.897 ± 0.0350.115 ± 0.1630.9963.243.62Fibrinogen α chainVQHIQLLQK√y7+0.963 ± 0.0180.057 ± 0.0860.9993.962.23Fibrinogen β chainEDGGGWWYNR√y4+1.000 ± 0.0130.047 ± 0.0621.0005.671.32Fibrinogen γ chainDNCCILDER√y4+1.022 ± 0.0180.014 ± 0.0860.9994.351.76Gelsolin, isoform 1TGAQELLRy4+0.984 ± 0.0110.028 ± 0.0431.0000.883.01Haptoglobin β chainVGYVSGWGRy6+1.049 ± 0.006−0.004 ± 0.0301.0001.050.78HemopexinNFPSPVDAAFRy7+0.989 ± 0.0130.030 ± 0.0601.0002.982.01Heparin cofactor IITLEAQLTPRy6+1.021 ± 0.0220.031 ± 0.1020.9992.042.30Inter-α-trypsin inhibitor HCAAISGENAGLVRy9+1.016 ± 0.0240.043 ± 0.1110.9993.402.07Kininogen-1YFIDFVAR√y6+0.965 ± 0.0100.065 ± 0.0481.0003.741.40l-selectincl-Selectin was excluded in calculation of the average values.AEIEYLEKy5+0.980 ± 0.1410.731 ± 0.5710.943-22.2Plasma retinol-binding proteinYWGVASFLQKy6+1.021 ± 0.0130.018 ± 0.0591.0002.974.17PlasminogenWELCDIPR√y6+1.000 ± 0.0120.038 ± 0.0571.0003.312.32ProthrombinYGFYTHVFR√y5+1.064 ± 0.0390.015 ± 0.1850.9963.034.49Serum amyloid P-componentVGEYSLYIGRy6+0.972 ± 0.0120.063 ± 0.0581.0001.812.16TransferrinEGYYGYTGAFR√y7+1.012 ± 0.0090.037 ± 0.0431.0002.860.94TransthyretinCPLMVK√y5+0.994 ± 0.0270.022 ± 0.1100.9981.903.32Vitamin d-binding proteinTHLPEVFLSKy3+0.951 ± 0.0140.014 ± 0.0640.9994.002.66VitronectinFEDGVLDPDYPRy5+0.961 ± 0.0270.097 ± 0.1270.9984.283.95Zinc-α2-glycoproteinEIPAWVPFDPAAQITKy10+0.946 ± 0.0170.064 ± 0.0810.9993.122.79Averagecl-Selectin was excluded in calculation of the average values.0.991 ± 0.0210.050 ± 0.0960.9993.792.67a CI, 95% confidence interval.b Accuracy was calculated based on the percentage of error relative to the known relative abundance ratios.c l-Selectin was excluded in calculation of the average values. Open table in a new tab Peptide samples were analyzed using either an Agilent 1100 LC system (Agilent Technologies) or ACQUITY UPLC (Waters) coupled on-line to a triple quadrupole mass spectrometer (TSQ Vantage; Thermo Fisher Scientific). The capillary analytical column was prepared by slurry-packing 3-μm Jupiter C18 bonded particles (Phen