Enriching the Phosphoproteome

The reversible phosphorylation of specific sites on proteins is implicated in the control of multiple cellular functions and processes, including cell growth and differentiation, cell death, gene expression, and signal transduction. Almost a third of all cellular proteins are believed to be phosphorylated at any given point in the cell cycle. Given the importance of protein phosphorylation in cell activities, characterization of the phosphoproteome will be key to understanding the mechanism of cellular processes and in identifying targets for therapeutic intervention. However, phosphoproteome analysis is a daunting analytical challenge. Phosphorylation is dynamic, with many proteins being phosphorylated at different sites and at different times. Therefore, phosphorylation at a given site can be substoichiometric and, thus, in very low abundance. Currently, the dominant technologies for characterizing protein phosphorylation are matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) and liquid chromatography (LC)–nanoelectrospray tandem MS. Most MS-based protein characterization is performed at the peptide level after cleavage with site-specific proteases. Phophorylation sites are less than ideal candidates for these approaches. Phosphorylated peptides do not ionize well in positive ion mode (the preferred approach in most cases), and are subject to ion suppression in the presence of the great excess of nonphosphorylated peptides.