Simultaneous Assessment of Kinetic, Site-Specific, and Structural Aspects of Enzymatic Protein Phosphorylation

摘要

Protein phosphorylation is a widespread process forming the mechanistic basis of'cellular signaling. Up to now, different aspects,for example, site-specificity, kinetics, role of co-factors,and sturcture-function relationships have been typically investigated by multiple techniques that are incom-patible with one another. The approach introduced here maximizs the amount of information gained on protein (complex)phosphorylation while minimizing sample han-dling,Using higy-resolution native mass spectrometry on intact prolein(assemblies up to 150 kD a we track the sequential incorporatino of phosphate groups and map their localization by peptide LC-MS/MS. On two model systems, the protein kinase G and the imterplay between Aurora kinase A and Bora, we demonstrate the simultaneous monitoring of various aspects of he phosphorylation process, namely the effect of different cofactors on PKG autophosphorylation and the interaction of AurA and Bora as both an enzyme-substrate pair and physical binding partners.