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

Philip Cohen, Bradford W. Gibson, Charles F.B. Holmes

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)153-168
Number of pages16
JournalMethods in Enzymology
Volume201
Issue numberC
DOIs
Publication statusPublished - 1991

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