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

Sasha A. Singh, Dominic Winter, Parizad M. Bilimoria, Azad Bonni, Hanno Steen, Judith A. Steen

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

Abstract

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 3Î 2 (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.

Original languageEnglish
Pages (from-to)504-508
Number of pages5
JournalNature Methods
Volume9
Issue number5
DOIs
StatePublished - May 2012

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