FLEXIQinase, a mass spectrometry-based assay, to unveil multikinase mechanisms.

We introduce a mass spectrometry-based method that provides residue-resolved quantitative information about protein phosphorylation. In this assay we combined our full-length expressed stable isotope-labeled protein for quantification strategy (FLEXIQuant) with a traditional kinase assay to determine the mechanisms of multikinase substrate phosphorylation such as priming-dependent kinase activities. The assay monitors the decrease in signal intensity of the substrate peptides and the concomitant increase in the (n x 80 Da)-shifted phosphorylated peptide. We analyzed the c-Jun N-terminal kinase (JNK)-dependent glycogen synthase kinase 3 beta (GSK3 beta) activity on doublecortin (DCX) revealing mechanistic details about the role of phosphorylation cross-talk in GSK3 beta activity and permitting an advanced model for GSK3 beta-mediated signaling.