Analysis of the in Vivo Phosphorylation States of Proteins by Fast Atom Bombardment Mass Spectrometry and Other Techniques

This chapter discusses the analysis of the in vivo phosphorylation states of proteins by fast atom bombardment mass spectrometry and other techniques. The most popular method for analyzing the in vivo phosphorylation state of a protein is to radiolabel it by incubating cells and tissues with ³²P-labeled inorganic phosphate. The cells/tissues are then lysed and the phosphoprotein is separated and identified by peptide mapping or immunoprecipitation. The chapter describes a new approach for the identification of in vivo phosphorylation sites and the measurement of phosphorylation stoichiometries. This approach uses a range of modern techniques and advances in protein chemistry antibodies. An alternative procedure is to determine the amount of inorganic phosphate in a protein or peptide directly, either by alkaline hydrolysis or by ashing in the presence of magnesium nitrate. This approach has been applied to tissues, such as skeletal muscle, which take up ³²P-labeled inorganic phosphate poorly and to proteins that can be isolated in large amounts.