Immunoaffinity profiling of tyrosine phosphorylation in cancer cells
Immunoaffinity profiling of tyrosine phosphorylation in cancer cells
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DOI：
10.1038/nbt1046
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发表时间：
2005-01-01
影响因子：
46.9
通讯作者：
Comb, MJ
中科院分区：
文献类型：
--
作者：
Rush, J;Moritz, A;Comb, MJ
酪氨酸激酶在人类癌症中起着重要作用，但驱动细胞增殖和存活的致癌信号传导途径一直难以识别，部分原因是途径的复杂性，部分原因是细胞酪氨酸磷酸化水平低。一般来说，全球磷酸蛋白质组学方法揭示了少量含有磷酸酪氨酸的肽。我们已经开发出一种策略，强调磷酸化蛋白质组的磷酸化酪氨酸成分，并确定了大量的酪氨酸磷酸化位点。用磷酸酪氨酸特异性抗体直接从蛋白酶消化的细胞蛋白提取物中分离含有磷酸酪氨酸的肽，并通过串联质谱法鉴定。将这种方法应用于几种细胞系统，包括癌细胞系，表明它可以用于识别活化的蛋白激酶及其磷酸化底物，而无需事先了解被激活的信号网络，这是分析正常和致癌信号网络的第一步。
Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.