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

Mol Cell Proteomics
Authors
Keywords
Abstract

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.

Year of Publication
2015
Journal
Mol Cell Proteomics
Volume
14
Issue
9
Pages
2375-93
Date Published
2015 Sep
ISSN
1535-9484
URL
DOI
10.1074/mcp.M114.046813
PubMed ID
25724909
PubMed Central ID
PMC4563722
Links
Grant list
U24 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