FLEXIQinase, a mass spectrometry-based assay, to unveil multi-kinase mechanisms

We introduce a mass spectrometry-based method that provides residue-resolved quantitative information about protein phosphorylation. In this FLEXIQinase 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 multi-kinase 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×80 Da)-shifted phosphorylated peptide. We analyzed the c-Jun N-terminal Kinase (JNK)-dependent glycogen synthase kinase 3β (GSK3β) activity on doublecortin (DCX) revealing mechanistic details about the role of phosphorylation cross-talk in GSK3β activity and permitting an advanced model for GSK3β-mediated signaling.