Quantitative Mass Spectrometric Multiple Reaction Monitoring Assays for Major Plasma Proteins

Quantitative LC-MS/MS assays were designed for tryptic peptides representing 53 high and medium abundance proteins in human plasma using a multiplexed multiple reaction monitoring (MRM) approach. Of these, 47 produced acceptable quantitative data, demonstrating within-run coefficients of variation (CVs) (n = 10) of 2–22% (78% of assays had CV <10%). A number of peptides gave CVs in the range 2–7% in five experiments (10 replicate runs each) continuously measuring 137 MRMs, demonstrating the precision achievable in complex digests. Depletion of six high abundance proteins by immunosubtraction significantly improved CVs compared with whole plasma, but analytes could be detected in both sample types. Replicate digest and depletion/digest runs yielded correlation coefficients (R2) of 0.995 and 0.989, respectively. Absolute analyte specificity for each peptide was demonstrated using MRM-triggered MS/MS scans. Reliable detection of L-selectin (measured at 0.67 μg/ml) indicates that proteins down to the μg/ml level can be quantitated in plasma with minimal sample preparation, yielding a dynamic range of 4.5 orders of magnitude in a single experiment. Peptide MRM measurements in plasma digests thus provide a rapid and specific assay platform for biomarker validation, one that can be extended to lower abundance proteins by enrichment of specific target peptides (stable isotope standards and capture by anti-peptide antibodies (SISCAPA)). Quantitative LC-MS/MS assays were designed for tryptic peptides representing 53 high and medium abundance proteins in human plasma using a multiplexed multiple reaction monitoring (MRM) approach. Of these, 47 produced acceptable quantitative data, demonstrating within-run coefficients of variation (CVs) (n = 10) of 2–22% (78% of assays had CV <10%). A number of peptides gave CVs in the range 2–7% in five experiments (10 replicate runs each) continuously measuring 137 MRMs, demonstrating the precision achievable in complex digests. Depletion of six high abundance proteins by immunosubtraction significantly improved CVs compared with whole plasma, but analytes could be detected in both sample types. Replicate digest and depletion/digest runs yielded correlation coefficients (R2) of 0.995 and 0.989, respectively. Absolute analyte specificity for each peptide was demonstrated using MRM-triggered MS/MS scans. Reliable detection of L-selectin (measured at 0.67 μg/ml) indicates that proteins down to the μg/ml level can be quantitated in plasma with minimal sample preparation, yielding a dynamic range of 4.5 orders of magnitude in a single experiment. Peptide MRM measurements in plasma digests thus provide a rapid and specific assay platform for biomarker validation, one that can be extended to lower abundance proteins by enrichment of specific target peptides (stable isotope standards and capture by anti-peptide antibodies (SISCAPA)). Accurate quantitation of proteins and peptides in plasma and serum is a challenging problem because of the complexity and extreme dynamic range that characterize these samples (1Anderson N.L. Anderson N.G. The human plasma proteome: history, character, and diagnostic prospects.Mol. Cell. Proteomics. 2002; 1: 845-867Abstract Full Text Full Text PDF PubMed Scopus (3564) Google Scholar). The widely adopted separative (survey) approach to proteomics, in which an attempt is made to detect all components, has proven to be limited in sensitivity toward low abundance proteins (2Anderson N.L. Polanski M. Pieper R. Gatlin T. Tirumalai R.S. Conrads T.P. Veenstra T.D. Adkins J.N. Pounds J.G. Fagan R. Lobley A. The human plasma proteome: a non-redundant list developed by combination of four separate sources.Mol. Cell. Proteomics. 2004; 3: 311-326Abstract Full Text Full Text PDF PubMed Scopus (760) Google Scholar) and typically provides limited quantitative precision. The alternative candidate-based approach, which relies on specific assays optimized for quantitative detection of selected proteins, can provide significantly increased sensitivity (into the pg/ml range) and precision (CVs 1The abbreviations used are: CV, coefficient of variation; QqQ, triple quadrupole; MRM, multiple reaction monitoring; S/N, signal-to-noise; Nat, natural sample-derived peptide; SIS, stable isotope-labeled internal standard; polySIS, polyprotein SIS; SISCAPA, stable isotope standards and capture by anti-peptide antibodies; MIDAS, multiple reaction monitoring-initiated detection and sequencing; MARS, multiple affinity removal system. < 5–10%) at the cost of restricting discovery potential toward novel proteins (3Kuhn E. Wu J. Karl J. Liao H. Zolg W. Guild B. Quantification of C-reactive protein in the serum of patients with rheumatoid arthritis using multiple reaction monitoring mass spectrometry and 13C-labeled peptide standards.Proteomics. 2004; 4: 1175-1186Crossref PubMed Scopus (370) Google Scholar). In practice, a combination of these approaches (one or more survey approaches for de novo biomarker discovery coupled with a candidate-based approach to biomarker validation in large sample sets) appears to provide an effective staged pipeline (4Anderson N.L. The roles of multiple proteomics platforms in a pipeline for new diagnostics.Mol. Cell. Proteomics. 4: Full Text Full Text PDF PubMed Scopus Google Scholar) for of plasma of and specific assays on the specificity of capture or detection to a specific antibodies can provide extreme specificity antibodies used in a and the of protein is in of assays multiplexed 2002; PubMed Scopus Google in Cell. Proteomics. 4: Full Text Full Text PDF PubMed Scopus Google Scholar) in in the of antibodies and in the of multiple assays in one spectrometry provides an alternative assay approach, on the of mass to a specific analyte and on measurements for In the of analytes R. T. T. spectrometry in PubMed Scopus Google and of a for the of in human serum using mass 2004; PubMed Scopus Google protein Quantitative of protein of and in and proteins by mass spectrometry multiple reaction PubMed Google and A. 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Quantification of C-reactive protein in the serum of patients with rheumatoid arthritis using multiple reaction monitoring mass spectrometry and 13C-labeled peptide standards.Proteomics. 2004; 4: 1175-1186Crossref PubMed Scopus (370) Google Scholar). In a specific tryptic peptide can be selected a of the protein which is and quantitated a internal stable isotope-labeled peptide J. Absolute of proteins and by A. PubMed Scopus Google to a of protein In an assay of the of the selected peptide and and an to the stable isotope-labeled C-reactive protein (3Kuhn E. Wu J. Karl J. Liao H. Zolg W. Guild B. Quantification of C-reactive protein in the serum of patients with rheumatoid arthritis using multiple reaction monitoring mass spectrometry and 13C-labeled peptide standards.Proteomics. 2004; 4: 1175-1186Crossref PubMed Scopus (370) Google of specific with PubMed Scopus Google human H. 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The roles of multiple proteomics platforms in a pipeline for new diagnostics.Mol. Cell. Proteomics. 4: Full Text Full Text PDF PubMed Scopus Google to tryptic peptides by in were of the peptides of produced acceptable in the of of tryptic peptides can be to in the used MS/MS data, in combination with to more target abundance peptides were detected in LC-MS/MS experiments in which a of high peptides in to abundance peptides were detected by of to all tryptic peptides a target protein and detected MRM by MS/MS in which MRM designed using a designed the typically more survey for detection of low abundance components, the approach to for more lower abundance peptides and the for MRM was by the combination of high sensitivity triple MRM and MS/MS on the triple mass A for MRM using these of is in in which the approach. 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Fagan R. Lobley A. The human plasma proteome: a non-redundant list developed by combination of four separate sources.Mol. Cell. Proteomics. 2004; 3: 311-326Abstract Full Text Full Text PDF PubMed Scopus (760) Google N.L. proteomics in the for of PubMed Scopus Google Scholar). that the protein in plasma, is by MRM in plasma in the that the dynamic range of proteins appears to be μg/ml to or dynamic range of orders of magnitude is with quantitation experiments using MRM on and the dynamic range of and proteins in human plasma (1Anderson N.L. Anderson N.G. The human plasma proteome: history, character, and diagnostic prospects.Mol. Cell. Proteomics. 2002; 1: 845-867Abstract Full Text Full Text PDF PubMed Scopus (3564) Google Scholar). 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