The catalytic and signaling activities of insulin receptor kinase （IRK） are regulated by the autophosphorylation of three tyrosine residues in a cytoplasmic protein-tyrosine kinase domain at Tyro 1158, Tyro 1162 and Tyro 1163. In this study, time-course of the auphosphorylation of the core kinase （residues 978-1283） from IRK was directly investigated by online electrospray ionization mass spectrometry. It is found that two tyrosine residues were phosphorylated in reaction time range of 30 min. This study implies that mass spectrometric technique must be a powerful tool to directly monitor the biological macromolecular modification and will also provide the information of the order and the mechanism of autophosphorylation at the tyrosine sites coupled with tandem mass spectrometric technique.
