Analysis of protein phosphorylation by mass spectrometry

Phosphorylation is one of the most frequently occurring post-translational modifications in proteins. In eukaryotic cells, protein phosphorylation on serine, threonine and tyrosine residues plays a crucial role as a modulator of protein function. A comprehensive analysis of protein phosphorylation involves the identification of the phosphoproteins, the exact localization of the residues that are phosphorylated and the quantitation of phosphorylation. In this short review we will summarize and discuss the methodologies currently available for the analysis and full characterization of phosphoproteins with emphasis on mass spectrometry-based techniques. In particular, we will discuss affinity-based purification of phosphopeptides coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI ToF-MS) analysis, their detection using mass mapping and precursor-ion scans, identification of modified sites by tandem mass spectrometry (MS/MS) and quantitative analysis.