Multiplexed, Quantitative Workflow for Sensitive Biomarker Discovery in Plasma Yields Novel Candidates for Early Myocardial Injury
Multiplexed, Quantitative Workflow for Sensitive Biomarker Discovery in Plasma Yields Novel Candidates for Early Myocardial Injury
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DOI：
10.1074/mcp.m114.046813
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发表时间：
2015-09-01
影响因子：
7
通讯作者：
Carr, Steven A.
中科院分区：
文献类型：
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作者：
Keshishian, Hasmik;Burgess, Michael W.;Carr, Steven A.
我们开发了一种新的血浆蛋白质分析平台，包括优化的样品制备、层析和MS分析方案。该工作流程利用化学等压质量标记对血浆蛋白质进行相对定量，实现了远比以前方法更深入的蛋白质组检测和定量，同时增加了样品吞吐量。我们将新的工作流程应用于肥厚型心肌病患者的血浆样本时间序列，这些患者正在接受治疗性的、“计划的”心肌梗死，这是一种独特的人类模型，每个人都充当自己的生物对照。在我们的实验中，我们信心十足地鉴定了超过5300种蛋白质，使用iTRAQ四链标记技术，每个样本平均鉴定出4600种蛋白质(每个蛋白质识别出两个或更多不同的多肽)。在所有16个患者样本中，近3400种蛋白质被量化。与以前发表的无标记方法相比，新方法对每个样本的蛋白质定量几乎增加了五倍，样本分析吞吐量增加了六到九倍。此外，这项研究提供了迄今为止可用的最大的高置信度血浆蛋白质组数据集。与无标记方法相比，相对定量的可靠性也大大提高，测量的iTRAQ比率和时间趋势与23-plex免疫MRM(IMRM)分析的结果很好地相关，该分析包含应用于相同患者样本的候选蛋白质的子集。改进的检测和定量的功能重要性反映在显着扩大的显著受调控的蛋白质列表中，这些蛋白质提供了许多新的候选生物标记蛋白。用TMT六链和十链试剂标记血浆样品的初步评估表明，即使进一步增加血浆分析的多重，相对于iTRAQ四链试剂，检测深度也不会有显著损失。利用我们的新平台获得的这些结果清楚地证明了在基于等离子体的生物标记物发现实验中使用等压质量标记试剂的价值。
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 multi-plexing 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.