Analysis of protein phosphorylation on a proteome-scale

Abstract

Phosphorylation, the most intensively studied and common PTM on proteins, is a complex biological phenomenon. Its complexity manifests itself in the large numbers of proteins that attach it, remove it and recognise it as a protein code. Since the first report of protein phosphorylation on vitellin 100 years ago, a wide variety of biochemical and analytical chemical approaches have been developed to enrich and detect protein phosphorylation. The last 5 years have witnessed a renaissance in methodologies capable of characterising protein phosphorylation on a proteome-scale. These technological advances have allowed identification of hundreds to thousands of phosphorylation sites in a proteome and have resulted in a profound paradigm shift. For the first time, using quantitative MS, the topology and significance of global phosphorylation networks may be investigated, marking a new era of cell signalling research. This review addresses recent technological advances in the purification of phosphorylated proteins and peptides and current MS-based strategies used to qualitatively and quantitatively probe these enriched phosphoproteomes. In addition, we review the application of complementary array-based technologies to derive signalling networks from kinase-substrate interactions and discuss future challenges in the field.