High-throughput and high-sensitivity phosphoproteomics with the EasyPhos platform
High-throughput and high-sensitivity phosphoproteomics with the EasyPhos platform
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DOI：
10.1038/s41596-018-0014-9
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发表时间：
2018-09-01
期刊：
影响因子：
14.8
通讯作者：
Mann, Matthias
中科院分区：
文献类型：
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作者：
Humphrey, Sean J.;Karayel, Ozge;Mann, Matthias
质谱分析通过实现对动态蛋白质磷酸化（“磷酸化蛋白质组学”）的全面研究，改变了细胞信号传导领域。近期的发展使得磷酸化蛋白质组学研究日益复杂，但实际挑战依然存在。EasyPhos工作流程解决了这些问题，并且足够简化，能够对数百个磷酸化蛋白质组进行分析，可定量的磷酸化位点深度超过10,000个。在此，我们介绍一种详细的更新工作流程，它进一步确保了在样本有限的条件下的高性能，同时还减少了样本制备时间。通过省去蛋白质沉淀步骤，并在单个96孔板中完成包括酶解在内的整个实验流程，我们现在极大地减少了样本损失和变异性的可能性。这使得重现性非常高，并且对样本量的要求很小（
Mass spectrometry has transformed the field of cell signaling by enabling global studies of dynamic protein phosphorylation ('phosphoproteomics'). Recent developments are enabling increasingly sophisticated phosphoproteomics studies, but practical challenges remain. The EasyPhos workflow addresses these and is sufficiently streamlined to enable the analysis of hundreds of phosphoproteomes at a depth of >10,000 quantified phosphorylation sites. Here we present a detailed and updated workflow that further ensures high performance in sample-limited conditions while also reducing sample preparation time. By eliminating protein precipitation steps and performing the entire protocol, including digestion, in a single 96-well plate, we now greatly minimize opportunities for sample loss and variability. This results in very high reproducibility and a small sample size requirement (