Label-Free Phosphoproteomic Approach for Kinase Signaling Analysis.
Label-Free Phosphoproteomic Approach for Kinase Signaling Analysis.
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用于激酶信号分析的无标记磷酸化蛋白质组学方法。
DOI：
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发表时间：
2017
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通讯作者：
P. Cutillas
中科院分区：
文献类型：
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作者：
E. Wilkes;P. Cutillas
磷酸化蛋白质组学是一个强大的平台，可以无偏见地分析激酶驱动的信号通路。磷酸化的定量可以通过标记或无标记质谱仪(MS)的方法来执行。由于其简单性和普适性，无标记方法在分子生物学研究中得到了广泛的接受和欢迎。然而，无标记定量磷酸化的分析工作流程需要克服几个障碍，才能使技术准确和精确。其中包括使用生化提取程序从复杂的多肽基质中高效和可重复地分离磷肽，以及可以处理被比较样品子集中丢失的MS/MS磷肽谱的分析策略。测试开发的工作流程的准确性是MS分析小分子的基本前提，这是通过构建校准曲线来证明每个分析物的定量线性来实现的。这种严谨的分析水平在使用基于标记或无标记技术的大规模蛋白质量化中很少表现出来。在这一章中，我们展示了一种不需要合成标准或标记蛋白质的方法来测试用液相色谱(LC)-MS定量的每一种磷酸多肽的线性。我们进一步描述了磷酸肽的可重复回收所需的适当的样品处理技术，并探索了能够处理丢失的MS/MS谱从而使无标记数据适合于此类分析的基本算法特征。本章中描述的组合技术将磷蛋白质组学的适用性扩展到以前不能用其他方法处理的问题。
Phosphoproteomics is a powerful platform for the unbiased profiling of kinase-driven signaling pathways. Quantitation of phosphorylation can be performed by means of either labeling or label-free mass spectrometry (MS) methods. Because of their simplicity and universality, label-free methodology is gaining acceptance and popularity in molecular biology research. Analytical workflows for label-free quantification of phosphorylation, however, need to overcome several hurdles for the technique to be accurate and precise. These include the use of biochemical extraction procedures that efficiently and reproducibly isolate phosphopeptides from complex peptide matrices and an analytical strategy that can cope with missing MS/MS phosphopeptide spectra in a subset of the samples being compared. Testing the accuracy of the developed workflows is an essential prerequisite in the analysis of small molecules by MS, and this is achieved by constructing calibration curves to demonstrate linearity of quantification for each analyte. This level of analytical rigor is rarely shown in large-scale quantification of proteins using either label-based or label-free techniques. In this chapter we show an approach to test linearity of quantification of each phosphopeptide quantified by liquid chromatography (LC)-MS without the need to synthesize standards or label proteins. We further describe the appropriate sample handling techniques required for the reproducible recovery of phosphopeptides and explore the essential algorithmic features that enable the handling of missing MS/MS spectra and thus make label-free data suitable for such analyses. The combined technology described in this chapter expands the applicability of phosphoproteomics to questions not previously tractable with other methodologies.