Amanchy Ramars, Kalume Dario E, Iwahori Akiko, Zhong Jun, Pandey Akhilesh
McKusick-Nathans Institute for Genetic Medicine and the Department of Biological Chemistry and Oncology, Johns Hopkins University, 733 N. Broadway, Baltimore, MD 21205, USA.
J Proteome Res. 2005 Sep-Oct;4(5):1661-71. doi: 10.1021/pr050134h.
Identification of phosphorylated proteins remains a difficult task despite technological advances in protein purification methods and mass spectrometry. Here, we report identification of tyrosine-phosphorylated proteins by coupling stable isotope labeling with amino acids in cell culture (SILAC) to mass spectrometry. We labeled HeLa cells with stable isotopes of tyrosine, or, a combination of arginine and lysine to identify tyrosine phosphorylated proteins. This allowed identification of 118 proteins, of which only 45 proteins were previously described as tyrosine-phosphorylated proteins. A total of 42 in vivo tyrosine phosphorylation sites were mapped, including 34 novel ones. We validated the phosphorylation status of a subset of novel proteins including cytoskeleton associated protein 1, breast cancer anti-estrogen resistance 3, chromosome 3 open reading frame 6, WW binding protein 2, Nice-4 and RNA binding motif protein 4. Our strategy can be used to identify potential kinase substrates without prior knowledge of the signaling pathways and can also be applied to profiling to specific kinases in cells. Because of its sensitivity and general applicability, our approach will be useful for investigating signaling pathways in a global fashion and for using phosphoproteomics for functional annotation of genomes.
尽管蛋白质纯化方法和质谱技术取得了进展，但磷酸化蛋白质的鉴定仍然是一项艰巨的任务。在此，我们报告通过将细胞培养中氨基酸的稳定同位素标记（SILAC）与质谱联用，鉴定酪氨酸磷酸化蛋白质。我们用酪氨酸的稳定同位素或精氨酸和赖氨酸的组合标记HeLa细胞，以鉴定酪氨酸磷酸化蛋白质。这使得能够鉴定出118种蛋白质，其中只有45种蛋白质先前被描述为酪氨酸磷酸化蛋白质。总共绘制了42个体内酪氨酸磷酸化位点，包括34个新位点。我们验证了包括细胞骨架相关蛋白1、乳腺癌抗雌激素抗性3、3号染色体开放阅读框6、WW结合蛋白2、Nice-4和RNA结合基序蛋白4在内的一部分新蛋白质的磷酸化状态。我们的策略可用于在不预先了解信号通路的情况下鉴定潜在的激酶底物，也可应用于细胞中特定激酶的分析。由于其敏感性和普遍适用性，我们的方法将有助于以全局方式研究信号通路，并将磷酸蛋白质组学用于基因组的功能注释。
