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

Nat Methods. 2012 Apr 8;9(5):504-8. doi: 10.1038/nmeth.1970.


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 × 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Doublecortin Domain Proteins
  • Glycogen Synthase Kinase 3 / chemistry
  • Glycogen Synthase Kinase 3 / metabolism*
  • JNK Mitogen-Activated Protein Kinases / chemistry
  • JNK Mitogen-Activated Protein Kinases / metabolism*
  • Microtubule-Associated Proteins / chemistry
  • Microtubule-Associated Proteins / metabolism*
  • Neuropeptides / chemistry
  • Neuropeptides / metabolism*
  • Phosphorylation
  • Signal Transduction
  • Tandem Mass Spectrometry / methods*


  • Doublecortin Domain Proteins
  • Microtubule-Associated Proteins
  • Neuropeptides
  • JNK Mitogen-Activated Protein Kinases
  • Glycogen Synthase Kinase 3