STRATEGIES FOR NONRADIOACTIVE METHODS IN THE LOCALIZATION OF PHOSPHORYLATED AMINO-ACIDS IN PROTEINS

Identification of O-phosphorylated amino acids within the primary stru cture of regulatory proteins is important in understanding the mechani sms by which their functions are regulated. In many cases radioactive labeling with [P-32]phosphate is tedious or sometimes impossible. Ther efore, we have established a series of new non-radioactive methods tha t permit the localization of phosphoserine, phosphothreonine, and phos photyrosine. After partial hydrolysis of a phosphopeptide or phosphopr otein, phosphoserine, phosphothreonine, or phosphotyrosine are determi ned by capillary electrophoresis as their dabsyl-derivatives. Chemical modification transforms phosphoserine or phosphothreonine to S-ethyl- cysteine or beta-methyl-S-ethyl-cysteine, respectively, allowing their localization during sequence analysis. We apply solid-phase sequencin g to overcome the limitations of the gas-phase sequenator in the case of phosphotyrosine-containing peptides. Liquid chromatography on-line connected to an electrospray mass spectrometer is a powerful new metho d of increasing importance in the protein chemistry field. It is espec ially well suited for identification of phosphoserine- or phosphothreo nine-containing peptides in a proteolytic digest of a phosphoprotein. In this article we will describe how to work with these new methods pr actically.