Quantitative phosphoproteomics for global profiling of kinase activity: a mass spectrometry-based activitomic approach to cell signaling.

摘要

We developed a novel strategy that coupled an in-vitro kinase assay using total cell lysates as enzyme substrate to quantitative proteomics to enable shotgun identification of phosphorylation events downstream of the protein kinase Akt1 and to quantify the affinity of this kinase towards ATP. In line with published data, motif analysis suggested that basophilic motifs (xxRxRxxSxxxxxxx/xxxxRxxSxxxxxxx) are required for high affinity (low Ac) utilization of ATP by Akt1. We also demonstrated the applicability of this approach to quantify endogenous kinase activities in an unbiased fashion and to amplify global kinase activity signals.