Zhang Guoan, Neubert Thomas A
Department of Pharmacology, New York University School of Medicine, New York, NY, USA.
Methods Mol Biol. 2009;527:79-92, xi. doi: 10.1007/978-1-60327-834-8_7.
In recent years, stable isotope labeling by amino acids in cell culture (SILAC) has become increasingly popular as a quantitative proteomic method. In SILAC experiments, proteins are metabolically labeled by culturing cells in media containing normal and heavy isotope amino acids. This makes proteins from the light and heavy cells distinguishable by mass spectrometry (MS) after the cell lysates are mixed and the proteins separated and/or enriched. SILAC is a powerful tool for the study of intracellular signal transduction. In particular, it has been very popular and successful in quantitative analysis of phosphotyrosine (pTyr) proteomes to characterize pTyr-dependent signaling pathways. In this chapter, we describe the SILAC procedure and use EphB signaling pathway as an example to illustrate the use of SILAC to investigate such pathways.
近年来，细胞培养中氨基酸稳定同位素标记法（SILAC）作为一种定量蛋白质组学方法越来越受欢迎。在SILAC实验中，通过在含有正常和重同位素氨基酸的培养基中培养细胞，对蛋白质进行代谢标记。当细胞裂解物混合后，蛋白质经分离和/或富集，然后通过质谱（MS）可区分来自轻细胞和重细胞的蛋白质。SILAC是研究细胞内信号转导的有力工具。特别是，它在磷酸酪氨酸（pTyr）蛋白质组的定量分析中非常流行且成功，用于表征pTyr依赖性信号通路。在本章中，我们描述了SILAC程序，并以EphB信号通路为例，说明如何使用SILAC来研究此类信号通路。
