Ke Mi, Chu Bizhu, Lin Lin, Tian Ruijun
Department of Chemistry, South University of Science and Technology of China, Shenzhen, 518055, People's Republic of China.
Materials Characterization and Preparation Center, South University of Science and Technology of China, Shenzhen, 518055, People's Republic of China.
Methods Mol Biol. 2017;1555:395-406. doi: 10.1007/978-1-4939-6762-9_22.
Dynamic tyrosine phosphorylation is a key molecular modulation for many signal transduction events. Because of their low abundance and dynamic nature in cells, the detection and enrichment of phosphotyrosine proteins has long relied on specific antibodies, such as 4G10 and P-Tyr-100. Another well-established approach for phosphotyrosine proteins recognition and enrichment is by their specific binding domains, such as Src homology 2 (SH2) domains. In this chapter, we describe a typical analytical approach for purifying specific SH2 domains, enriching specific phosphotyrosine proteins from activated cells, mass spectrometry analysis, and related data analysis.
动态酪氨酸磷酸化是许多信号转导事件的关键分子调节方式。由于其在细胞中的丰度较低且具有动态特性，磷酸化酪氨酸蛋白的检测和富集长期以来依赖于特异性抗体，如4G10和P-Tyr-100。另一种成熟的磷酸化酪氨酸蛋白识别和富集方法是通过其特异性结合结构域，如Src同源2（SH2）结构域。在本章中，我们描述了一种典型的分析方法，用于纯化特定的SH2结构域、从活化细胞中富集特定的磷酸化酪氨酸蛋白、进行质谱分析以及相关的数据分析。
