A site-specific, multiplexed kinase activity assay using stable-isotope dilution and high-resolution mass spectrometry.

ABSTRACT: Most kinases are capable of recognizing and phosphorylating peptides containing short, linear sequence motifs. To measure the activation state of many kinases from the same cell lysate, we created a multiplexed, mass-spectrometry-based in vitro kinase assay. Ninety chemically synthesized peptides derived from well-characterized peptide substrates and in vivo phosphorylation sites with either known or previously unidentified upstream kinases were reacted individually in a plate format with crude cell lysates and ATP. Phosphorylation rates were directly measured based on the addition of 90 same-sequence, site-specific phosphopeptides enriched in stable isotopes to act as ideal quantitative internal standards for analysis by liquid chromatography coupled to tandem mass spectrometry. This appr

SUBMITTER: Yu Y 

PROVIDER: S-EPMC2710625 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature