Combined enzymatic and data mining approaches for comprehensive phosphoproteome analyses
Combined enzymatic and data mining approaches for comprehensive phosphoproteome analyses
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DOI：
10.1074/mcp.m700383-mcp200
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发表时间：
2008-04-01
影响因子：
7
通讯作者：
Thibault, Pierre
中科院分区：
文献类型：
--
作者：
Marcantonio, Maria;Trost, Matthias;Thibault, Pierre
蛋白质磷酸化是一种中枢细胞信号事件，是一系列重要生理过程的基础。亲和层析和质谱的进步现在提供了同时鉴定和定量数千个磷酸化位点的能力。鉴于磷酸肽的抑制作用和MS/MS光谱的可变质量，全面的磷酸化蛋白质组分析提出了相当大的分析挑战。这项工作提出了一个综合的酶和数据挖掘的方法，使全面检测的本地和假定的磷酸肽碱性磷酸酶消化二氧化钛（TiO 2）富集的细胞提取物。从J774巨噬细胞提取物的750多个磷酸和脱磷酸肽对的保留时间的相关性表明，去除磷酸基团可以赋予的增益或损失的疏水性，这部分是由形成的盐桥与近端氨基。去磷酸化还导致MS灵敏度平均增加2倍，这有利于肽测序。更重要的是，碱性磷酸酶消化增强了从TiO 2富集的细胞提取物中推定的磷酸肽的总体群体，提供了一种独特的方法来分析多磷酸化同源物，否则这些同源物将无法检测到。这种方法的应用程序被证明为差分磷酸化蛋白质组分析的小鼠巨噬细胞暴露于干扰素-γ 5分钟。二氧化钛富集使识别1143磷酸肽从432种不同的蛋白质，其中125磷酸肽显示2倍的变化后，干扰素-γ曝光。碱性磷酸酶的使用几乎使假定的磷酸肽分配的数量增加了一倍，从而观察到参与囊泡运输、活性氧产生和mRNA翻译的关键干扰素-γ信号传导事件。
Protein phosphorylation is a central cell signaling event that underlies a broad spectrum of key physiological processes. Advances in affinity chromatography and mass spectrometry are now providing the ability to identify and quantitate thousands of phosphorylation sites simultaneously. Comprehensive phosphoproteome analyses present sizable analytical challenges in view of suppression effects of phosphopeptides and the variable quality of MS/MS spectra. This work presents an integrated enzymatic and data mining approach enabling the comprehensive detection of native and putative phosphopeptides following alkaline phosphatase digestion of titanium dioxide (TiO2)-enriched cell extracts. The correlation of retention times of more than 750 phospho- and dephosphopeptide pairs from J774 macrophage cell extracts indicated that removal of the phosphate groups can impart a gain or a loss in hydrophobicity that is partly explained by the formation of a salt bridge with proximal amino groups. Dephosphorylation also led to an average 2-fold increase in MS sensitivity that facilitated peptide sequencing. More importantly, alkaline phosphatase digestion enhanced the overall population of putative phosphopeptides from TiO2-enriched cell extracts providing a unique approach to profile multiphosphorylated cognates that would have remained otherwise undetected. The application of this approach is demonstrated for differential phosphoproteome analyses of mouse macrophages exposed to interferon-gamma for 5 min. TiO2 enrichment enabled the identification of 1143 phosphopeptides from 432 different proteins of which 125 phosphopeptides showed a 2-fold change upon interferon-gamma exposure. The use of alkaline phosphatase nearly doubled the number of putative phosphopeptides assignments leading to the observation of key interferon-gamma signaling events involved in vesicle trafficking, production of reactive oxygen species, and mRNA translation.