Serine/Threonine Phosphorylation Site Identification by ESI-MS

INTRODUCTIONMuch of the protein phosphorylation within cells is mediated by the activity of independent kinases. However, autophosphorylation of proteins is another way signal transduction pathways are regulated. This protocol is designed to distinguish between autophosphorylation and endogenous phosphorylation. For the localization of all endogenous phosphorylation sites, the protein is digested with trypsin and the peptides are analyzed by on-line coupling of nano-HPLC and ESI-MS. To determine the autophosphorylation sites, the protein is incubated with radioactive ATP prior to digestion with trypsin. Only 20 pmol of protein is used for each experiment. The stoichiometry of phosphorylated protein is below 5% of the total protein. Prior to analysis, the protein is separated by 10% SDS-PAGE and the protein band is excised. Before digestion, the protein is reduced and alkylated in both cases.