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

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.

SUBMITTER: Singh SA

PROVIDER: S-EPMC3595540 | biostudies-other | 2012 Apr

REPOSITORIES: biostudies-other

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

Singh Sasha A SA Winter Dominic D Bilimoria Parizad M PM Bonni Azad A Steen Hanno H Steen Judith A JA

Nature methods 20120408 5

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 ...[more]

PMID: 22484849

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