Use of stable isotope labeling by amino acids in cell culture (SILAC) for phosphotyrosine protein identification and quantitation.

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.