Measurement of Protein Phosphorylation Stoichiometry by Selected Reaction Monitoring Mass Spectrometry
Measurement of Protein Phosphorylation Stoichiometry by Selected Reaction Monitoring Mass Spectrometry
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DOI：
10.1021/pr100024a
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发表时间：
2010-05-01
影响因子：
4.4
通讯作者：
Moran, Michael F.
中科院分区：
文献类型：
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作者：
Jin, Lily L.;Tong, Jiefei;Moran, Michael F.
蛋白质磷酸化在特定氨基酸位点的化学计量可用于推断修饰的意义及其在细胞中的生物学功能。然而，组织中磷酸化化学计量的检测和定量仍然是一个重大挑战。在这里，我们描述了一种高灵敏度，无标记定量蛋白磷酸化化学计量的策略。方法开发包括分析合成肽，以确定与同源肽/磷酸肽对的质谱信号相关的常数，以及通过使用高分辨率傅里叶变换质谱法（FTMS）和选择反应监测质谱法（SRM）检测同源肽。通过FTMS分析提取的离子电流，在转染的人HEK 293 T细胞和两个培养的人多发性骨髓瘤株中测定蛋白酪氨酸激酶林恩中的两个酪氨酸残基（酪氨酸-194和酪氨酸-397）的磷酸化化学计量。为了实现测量组织中磷酸化化学计量的高灵敏度，开发了SRM方法并将其应用于分析多发性骨髓瘤异种移植肿瘤中林恩磷酸化位点的磷酸化化学计量。Western免疫印迹法用于验证质谱结果。SRM方法在分析临床样品中具有潜在的应用，其中蛋白磷酸化化学计量学可能代表重要的药效学生物标志物。
The stoichiometry of protein phosphorylation at specific amino acid sites may be used to infer on the significance of the modification, and its biological function in the cell. However, detection and quantification of phosphorylation stoichiometry in tissue remain a significant challenge. Here we describe a strategy for highly sensitive, label-free quantification of protein phosphorylation stoichiometry. Method development included the analysis of synthetic peptides in order to determine constants to relate the mass spectrometry signals of cognate peptide/phosphopeptide pairs, and the detection of the cognate peptides by using high resolution Fourier Transform mass spectrometry (FTMS) and selected reaction monitoring mass spectrometry (SRM). By analyzing extracted ion currents by FTMS, the phosphorylation stoichiometries of two tyrosine residues (tyrosine-194 and tyrosine-397) in the protein tyrosine kinase Lyn were determined in transfected human HEK293T cells and two cultured human multiple myeloma strains. To achieve high sensitivity to measure phosphorylation stoichiometry in tissue, SRM methods were developed and applied for the analysis of phosphorylation stoichiometries of Lyn phospho-sites in multiple myeloma xenograft tumors. Western immuno-blotting was used to verify mass spectrometry findings. The SRM method has potential applications in analyzing clinical samples wherein protein phosphorylation stoichiometries may represent important pharmacodynamic biomarkers.