Strategy for SRM-based Verification of Biomarker Candidates Discovered by iTRAQ Method in Limited Breast Cancer Tissue Samples
Strategy for SRM-based Verification of Biomarker Candidates Discovered by iTRAQ Method in Limited Breast Cancer Tissue Samples
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DOI：
10.1021/pr300322q
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发表时间：
2012-08-01
影响因子：
4.4
通讯作者：
Tomonaga, Takeshi
中科院分区：
文献类型：
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作者：
Muraoka, Satoshi;Kume, Hideaki;Tomonaga, Takeshi
由于基于LC-MS的定量蛋白质组学在过去十年中越来越多地应用于广泛的生物学应用，许多研究已经在大量蛋白质组中进行了相对和/或绝对丰度测定。在这项研究中，我们发现预后生物标志物候选人从有限的乳腺癌组织样本使用发现通过验证策略结合iTRAQ方法，然后选择反应监测/多反应监测分析（SRM/MRM）。我们在18个患者组织样本中以高置信度鉴定并定量了5122种蛋白质（合并高风险（n = 9）或低风险（n = 9））。基因本体分析表明，共2480个蛋白质（48.4%）被标注为膜蛋白，16.1%为质膜蛋白，6.6%为胞外蛋白。选择两组中具有>2倍差异的49种蛋白质用于进一步分析，并使用SRM/MRM在16个个体组织样品（高风险（n = 9）或低风险（n = 7））中进行验证。两组中有23个蛋白质差异表达，其中MFAP 4和GP 2在17个组织样本（高风险（n = 9）或低风险（n = 8））中进一步通过Western印迹和24个组织样本（高风险（n = 12）或低风险（n = 12））中的免疫组织化学（IHC）证实。这些结果表明，iTRAQ和SRM/MRM蛋白质组学的组合将是一个强大的工具，用于识别和验证候选蛋白质生物标志物。
Since LC-MS-based quantitative proteomics has become increasingly applied to a wide range of biological applications over the past decade, numerous studies have performed relative and/or absolute abundance determinations across large sets of proteins. In this study, we discovered prognostic biomarker candidates from limited breast cancer tissue samples using discovery-through-verification strategy combining iTRAQ method followed by selected reaction monitoring/multiple reaction monitoring analysis (SRM/MRM). We identified and quantified 5122 proteins with high confidence in 18 patient tissue samples (pooled high-risk (n = 9) or low-risk (n = 9)). A total of 2480 proteins (48.4%) of them were annotated as membrane proteins, 16.1% were plasma membrane and 6.6% were extracellular space proteins by Gene Ontology analysis. Forty-nine proteins with >2-fold differences in two groups were chosen for further analysis and verified in 16 individual tissue samples (high-risk (n = 9) or low-risk (n = 7)) using SRM/MRM. Twenty-three proteins were differentially expressed among two groups of which MFAP4 and GP2 were further confirmed by Western blotting in 17 tissue samples (high-risk (n = 9) or low-risk (n = 8)) and Immunohistochemistry (IHC) in 24 tissue samples (high-risk (n = 12) or low-risk (n = 12)). These results indicate that the combination of iTRAQ and SRM/MRM proteomics will be a powerful tool for identification and verification of candidate protein biomarkers.