UNIT 24.4 Phosphoproteomics

Abstract

Protein phosphorylation is one of the most important mechanisms of regulating protein function in cells, and it plays an important role in controlling diverse biological processes, including cellular proliferation, migration, and metabolism. The term “phosphoproteome” refers to the complement of proteins that undergoes phosphorylation, the extent of their phosphorylation status at the level of individual residues, as well as the dynamics of the phosphorylation events in response to various stimuli. This unit provides methods for enrichment of phosphorylated proteins and peptides using anti-phosphotyrosine antibodies or titanium dioxide, respectively. Support protocols are provided for two detergent-free cell lysis methods, fractionation of proteins prior to enrichment, and use of stable isotope labeling by amino acids in cell culture (SILAC) method for studying dynamics of phosphorylation events. Curr. Protoc. Protein Sci. 50:24.4.1-24.4.21. © 2007 by John Wiley & Sons, Inc.