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Mol Cell Proteomics DOI:10.1074/mcp.M900140-MCP200

Quantification of cardiovascular biomarkers in patient plasma by targeted mass spectrometry and stable isotope dilution.

Publication TypeJournal Article
Year of Publication2009
AuthorsKeshishian, H, Addona, T, Burgess, M, Mani, DR, Shi, X, Kuhn, E, Sabatine, MS, Gerszten, RE, Carr, SA
JournalMol Cell Proteomics
Volume8
Issue10
Pages2339-49
Date Published2009 Oct
ISSN1535-9484
KeywordsAmino Acid Sequence, Biological Assay, Biomarkers, Cardiovascular Diseases, Humans, Indicator Dilution Techniques, Isotope Labeling, Mass Spectrometry, Molecular Sequence Data, Myocardium, Peptides, Proteins
Abstract

Verification of candidate biomarkers requires specific assays to selectively detect and quantify target proteins in accessible biofluids. The primary objective of verification is to screen potential biomarkers to ensure that only the highest quality candidates from the discovery phase are taken forward into preclinical validation. Because antibody reagents for a clinical grade immunoassay often exist for a small number of candidates, alternative methodologies are required to credential new and unproven candidates in a statistically viable number of serum or plasma samples. Using multiple reaction monitoring coupled with stable isotope dilution MS, we developed quantitative, multiplexed assays in plasma for six proteins of clinical relevance to cardiac injury. The process described does not require antibodies for immunoaffinity enrichment of either proteins or peptides. Limits of detection and quantitation for each signature peptide used as surrogates for the target proteins were determined by the method of standard addition using synthetic peptides and plasma from a healthy donor. Limits of quantitation ranged from 2 to 15 ng/ml for most of the target proteins. Quantitative measurements were obtained for one to two signature peptides derived from each target protein, including low abundance protein markers of cardiac injury in the nanogram/milliliter range such as the cardiac troponins. Intra- and interassay coefficients of variation were predominantly

URLhttp://www.mcponline.org/cgi/pmidlookup?view=long&pmid=19596694
DOI10.1074/mcp.M900140-MCP200
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/19596694?dopt=Abstract

Alternate JournalMol. Cell Proteomics
PubMed ID19596694
PubMed Central IDPMC2758760
Grant ListU01 HL081341 / HL / NHLBI NIH HHS / United States
U24 CA126476 / CA / NCI NIH HHS / United States
1U24 CA126476-02 / CA / NCI NIH HHS / United States
U01-HL081341 / HL / NHLBI NIH HHS / United States