McLaughlin Centre for Molecular Medicine, MaRS East Tower, 101 College Street, Toronto, M5G 1L7, Canada.
J Proteome Res. 2010 May 7;9(5):2752-61. doi: 10.1021/pr100024a.
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.
蛋白质在特定氨基酸位点磷酸化的化学计量可能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，我们描述了一种用于高度敏感、无标记定量测定蛋白质磷酸化化学计量的策略。方法开发包括分析合成肽，以确定将同源肽/磷酸肽对的质谱信号相关联的常数，并使用高分辨率傅里叶变换质谱（FTMS）和选择反应监测质谱（SRM）检测同源肽。通过分析 FTMS 的提取离子电流，在转染的人 HEK293T 细胞和两种培养的人多发性骨髓瘤株中测定了蛋白酪氨酸激酶 Lyn 中两个酪氨酸残基（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，并将其应用于多发性骨髓瘤异种移植肿瘤中 Lyn 磷酸化位点的磷酸化化学计量分析。Western 免疫印迹用于验证质谱发现。SRM 方法在分析可能代表重要药效动力学生物标志物的临床样本方面具有潜在应用。
