Kang Un-Beom, Alexander William M, Marto Jarrod A
Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA.
Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA, USA.
Proteomics. 2017 Mar;17(6). doi: 10.1002/pmic.201600437.
Postgenomic studies continue to highlight the potential clinical importance of protein phosphorylation signaling pathways in drug discovery. Unfortunately, the dynamic range and variable stoichiometry of protein phosphorylation continues to stymie efforts to achieve comprehensive characterization of the human phosphoproteome. In this study, we develop a complementary, two-stage method for enrichment of cysteine-containing phosphopeptides combined with TMT multiplex labeling for relative quantification. The use of this approach with multidimensional fractionation in mammalian cells yielded more than 7000 unique cys-phosphopeptide sequences, comprising 15-20% novel phosphorylation sites. The use of our approach in combination with pharmacologic inhibitors of the mechanistic target of rapamycin complex 1 and 2 identified several putatively novel protein substrates for the mechanistic target of rapamycin kinase.
后基因组学研究不断凸显蛋白质磷酸化信号通路在药物发现中的潜在临床重要性。遗憾的是，蛋白质磷酸化的动态范围和可变化学计量比仍阻碍着对人类磷酸化蛋白质组进行全面表征的努力。在本研究中，我们开发了一种互补的两阶段方法，用于富集含半胱氨酸的磷酸肽，并结合TMT多重标记进行相对定量。在哺乳动物细胞中使用这种方法结合多维分级分离，产生了7000多个独特的半胱氨酸磷酸肽序列，其中包含15 - 20%的新磷酸化位点。将我们的方法与雷帕霉素复合物1和2的机制靶点的药理抑制剂相结合，鉴定出了几种雷帕霉素激酶机制靶点的潜在新蛋白质底物。
