Phosphoproteome analysis of HeLa cells using stable isotope labeling with amino acids in cell culture (SILAC)
Phosphoproteome analysis of HeLa cells using stable isotope labeling with amino acids in cell culture (SILAC)
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DOI：
10.1021/pr050134h
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发表时间：
2005-09-01
影响因子：
4.4
通讯作者：
Pandey, A
中科院分区：
文献类型：
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作者：
Amanchy, R;Kalume, DE;Pandey, A
尽管蛋白质纯化方法和质谱技术取得了进展，但磷酸化蛋白的鉴定仍然是一项艰巨的任务。在这里，我们报告的酪氨酸磷酸化蛋白质的鉴定耦合稳定同位素标记与氨基酸在细胞培养（SILAC）质谱。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。这允许鉴定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。共绘制了42个体内酪氨酸磷酸化位点，其中包括34个新位点。我们验证了包括细胞骨架相关蛋白1、乳腺癌抗雌激素抵抗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在内的一组新蛋白的磷酸化状态。我们的策略可用于识别潜在的激酶底物，而无需事先了解信号通路，也可用于分析细胞中的特定激酶。由于其敏感性和普遍适用性，我们的方法将是有用的，在全球范围内调查信号通路，并使用磷酸化蛋白质组学基因组的功能注释。
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.