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Mol Cell Proteomics DOI:10.1074/mcp.M114.046813

Multiplexed, Quantitative Workflow for Sensitive Biomarker Discovery in Plasma Yields Novel Candidates for Early Myocardial Injury.

Publication TypeJournal Article
Year of Publication2015
AuthorsKeshishian, H, Burgess, MW, Gillette, MA, Mertins, P, Clauser, KR, Mani, DR, Kuhn, EW, Farrell, LA, Gerszten, RE, Carr, SA
JournalMol Cell Proteomics
Date Published2015 Sep
KeywordsBiomarkers, Cardiomyopathy, Hypertrophic, Gas Chromatography-Mass Spectrometry, Humans, Interrupted Time Series Analysis, Myocardial Infarction, Proteomics, Reproducibility of Results, Workflow

We have developed a novel plasma protein analysis platform with optimized sample preparation, chromatography, and MS analysis protocols. The workflow, which utilizes chemical isobaric mass tag labeling for relative quantification of plasma proteins, achieves far greater depth of proteome detection and quantification while simultaneously having increased sample throughput than prior methods. We applied the new workflow to a time series of plasma samples from patients undergoing a therapeutic, "planned" myocardial infarction for hypertrophic cardiomyopathy, a unique human model in which each person serves as their own biologic control. Over 5300 proteins were confidently identified in our experiments with an average of 4600 proteins identified per sample (with two or more distinct peptides identified per protein) using iTRAQ four-plex labeling. Nearly 3400 proteins were quantified in common across all 16 patient samples. Compared with a previously published label-free approach, the new method quantified almost fivefold more proteins/sample and provided a six- to nine-fold increase in sample analysis throughput. Moreover, this study provides the largest high-confidence plasma proteome dataset available to date. The reliability of relative quantification was also greatly improved relative to the label-free approach, with measured iTRAQ ratios and temporal trends correlating well with results from a 23-plex immunoMRM (iMRM) assay containing a subset of the candidate proteins applied to the same patient samples. The functional importance of improved detection and quantification was reflected in a markedly expanded list of significantly regulated proteins that provided many new candidate biomarker proteins. Preliminary evaluation of plasma sample labeling with TMT six-plex and ten-plex reagents suggests that even further increases in multiplexing of plasma analysis are practically achievable without significant losses in depth of detection relative to iTRAQ four-plex. These results obtained with our novel platform provide clear demonstration of the value of using isobaric mass tag reagents in plasma-based biomarker discovery experiments.


Alternate JournalMol. Cell Proteomics
PubMed ID25724909
PubMed Central IDPMC4563722
Grant ListU24 CA160034 / CA / NCI NIH HHS / United States
R01HL096738 / HL / NHLBI NIH HHS / United States
R01 HL096738 / HL / NHLBI NIH HHS / United States
HHSN268201000033C / / PHS HHS / United States
U01 CA152990 / CA / NCI NIH HHS / United States
HHSN268201000033C / HL / NHLBI NIH HHS / United States
U24CA160034 / CA / NCI NIH HHS / United States
5U01CA152990-05 / CA / NCI NIH HHS / United States