Advances in protein phosphorylation analysis by capillary zone electrophoresis with online microreactor coupled with mass spectrometry

While reversible protein phosphorylation plays an important role in many cellular processes, simple and reliable measurement of the stoichiometry of phosphorylation can be challenging. This measurement is confounded by differences in the ionization efficiency of phosphorylated and unphosphorylated sites during analysis by mass spectrometry. Diagonal capillary electrophoresis is a two-dimensional separation method that incorporates an immobilized alkaline phosphatase microreactor at the distal end of the first capillary and employs identical electrophoretic separation modes in both dimensions. By online dephosphorylation in the capillary, there is no bias in ionization efficiency, and phosphorylation stoichiometry can be determined by the ratio of the signal of the two forms. In the work, I demonstrate diagonal capillary electrophoresismass spectrometry for characterization of protein phosphorylation, the accurate determination of this stoichiometry and applied this technique in the study of kinase activities.