A Strategy for Large-Scale Phosphoproteomics and SRM-Based Validation of Human Breast Cancer Tissue Samples
A Strategy for Large-Scale Phosphoproteomics and SRM-Based Validation of Human Breast Cancer Tissue Samples
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DOI：
10.1021/pr3005474
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发表时间：
2012-11-01
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：
Narumi, Ryohei;Murakami, Tatsuo;Tomonaga, Takeshi
蛋白质磷酸化是 A.细胞信号传导途径的关键机制和异常磷酸化与许多人类疾病有关。因此，磷酸蛋白质组学方法可以有助于识别关键生物标志物，以评估疾病发病机制和药物靶点。此外，对大规模磷酸化蛋白质组分析的仔细验证（当前基于蛋白质的生物标志物发现中所缺乏的）显着增加了已识别生物标志物的价值。在这里，我们使用 IMAC 结合同量异位标签进行相对定量 (iTRAQ) 技术进行大规模差异磷酸化蛋白质组分析，并通过选择/多重反应监测 (SRM/MRM) 对高风险和低风险复发组的人类乳腺癌组织进行后续验证。我们在 3401 个蛋白质上鉴定了 8309 个磷酸化位点，其中 3766 个磷酸肽（1927 个磷蛋白）能够被定量，并且 133 个磷酸肽（117 个磷蛋白）在两组之间存在差异表达。其中，选择了19种磷酸肽进行进一步验证，并以稳定同位素肽作为参考，通过SRM成功定量了15种。通过 SRM 量化的高风险组和低风险组之间的磷酸肽比率与基于 iTRAQ 的量化有很好的相关性，但有一些例外。这些结果表明，大规模磷酸化蛋白质组定量与基于 SRM 的验证相结合是使用临床样本发现生物标志物的强大工具。
Protein phosphorylation is a. key mechanism of cellular signaling pathways and aberrant phosphorylation has been implicated in a number of human diseases. Thus, approaches in phosphoproteomics can contribute to the identification of key biomarkers to assess disease pathogenesis and drug targets. Moreover, careful validation of large-scale phosphoproteome analysis, which is lacking in the current protein-based biomarker discovery, significantly increases the value of identified biomarkers. Here, we performed large-scale differential phosphoproteome analysis using IMAC coupled with the isobaric tag for relative quantification (iTRAQ) technique and subsequent validation by selected/multiple reaction monitoring (SRM/MRM) of human breast cancer tissues in high- and low-risk recurrence groups. We identified 8309 phosphorylation sites on 3401 proteins, of which 3766 phosphopeptides (1927 phosphoproteins) were able to be quantified and 133 phosphopeptides (117 phosphoproteins) were differentially expressed between the two groups. Among them, 19 phosphopeptides were selected for further verification and 15 were successfully quantified by SRM using stable isotope peptides as a reference. The ratio of phosphopeptides between high- and low risk groups quantified by SRM was well correlated with iTRAQ:based quantification with a few exceptions. These results suggest that large-scale phosphoproteome quantification coupled with SRM-based validation is a powerful tool for biomarker discovery using clinical samples.