학술논문
FLEXIQinase, a mass spectrometry-based assay, to unveil multikinase mechanisms.
Document Type
Article
Author
Source
Subject
*MASS spectrometry
*BIOLOGICAL assay
*C-Jun N-terminal kinases
*GLYCOGEN synthase kinase-3
*PHOSPHORYLATION
*PEPTIDES
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Language
ISSN
1548-7091
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 3? (GSK3?) activity on doublecortin (DCX) revealing mechanistic details about the role of phosphorylation cross-talk in GSK3? activity and permitting an advanced model for GSK3?-mediated signaling. [ABSTRACT FROM AUTHOR]