SILAC-Based Quantitative Phosphoproteomics in Yeast

Abstract

In this chapter, detailed procedures for stable isotope labeling with amino acids in cell culture, SILAC labeling of yeast auxotroph, optimization and evaluation of phosphopeptide enrichment, and sample preparation and analysis by high-resolution LC-MS/M, identification of phosphosites, and quantification methods are described. We report methods for the application of double SILAC to yeast using a combination of labeled lysine and labeled arginine. The combination of SILAC-based quantitation with phosphopeptides enrichment by TiO2 in a batch that enables measurement of protein posttranslational modifications is a powerful application to analyze the global phosphoproteome for studies in signaling pathways.