Deep Profiling of Proteome and Phosphoproteome by Isobaric Labeling, Extensive Liquid Chromatography, and Mass Spectrometry.
Deep Profiling of Proteome and Phosphoproteome by Isobaric Labeling, Extensive Liquid Chromatography, and Mass Spectrometry.
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DOI：
10.1016/bs.mie.2016.10.007
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发表时间：
2017
影响因子：
--
通讯作者：
Peng J
中科院分区：
文献类型：
--
作者：
Bai B;Tan H;Pagala VR;High AA;Ichhaporia VP;Hendershot L;Peng J
基于质谱的蛋白质组学在蛋白质和翻译后修饰的综合分析方面经历了前所未有的进步，特别是液相色谱-串联质谱（LC-MS/MS）和同量异位素标记多重能力的技术进步。在这里，我们介绍了一种深度蛋白质组学分析方案，该方案将10重串联质量标签（TMT）标记与优化的LC-MS/MS平台相结合，以定量整个蛋白质组和磷酸化蛋白质组。主要步骤包括蛋白质提取和消化、TMT标记、二维液相色谱、TiO 2介导的磷酸肽富集、高分辨质谱和计算机数据处理。该方案常规导致哺乳动物样品中超过10，000种蛋白质和大约30，000种磷酸化位点的可靠定量。实施质量控制步骤以排除故障和评估实验变异。这种多路复用的稳健方法提供了一种强大的工具，用于在系统水平上剖析各种复杂样品中的蛋白质组特征，范围从细胞培养物、动物组织到人类临床标本。
Mass spectrometry-based proteomics has experienced an unprecedented advance in comprehensive analysis of proteins and posttranslational modifications, with particular technical progress in liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here we introduce a deep proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an optimized LC-MS/MS platform to quantitate whole proteome and phosphoproteome. The major steps include protein extraction and digestion, TMT labeling, two dimensional liquid chromatography, TiO2-mediated phosphopeptide enrichment, high resolution mass spectrometry, and computational data processing. This protocol routinely leads to confident quantification of more than 10,000 proteins and approximately 30,000 phosphosites in mammalian samples. Quality control steps are implemented for troubleshooting and evaluating experimental variation. Such a multiplexed robust method provides a powerful tool for dissecting proteomic signatures at the systems level in a variety of complex samples, ranging from cell culture, animal tissues to human clinical specimens.