Analysis of phosphorylated proteins and peptides by mass spectrometry

Phosphorylation on serine, threonine and tyrosine residues is an extremely important modulator of protein function. Therefore, there is a great need f or methods capable of accurately elucidating sites of phosphorylation. Alth ough full characterization of phosphoproteins remains a formidable analytic al challenge, mass spectrometry has emerged as an increasingly viable tool for this task. This review summarizes the methodologies currently available for the analysis of phosphoproteins by mass spectrometry, including enrich ment of compounds of interest using immobilized metal affinity chromatograp hy and chemical tagging techniques, detection of phosphopeptides using mass mapping and precursor ion scans, localization of phosphorylation sites by peptide sequencing, and quantitation of phosphorylation by the introduction of mass tags. Despite the variety of powerful analytical methods that are now available, complete characterization of the phosphorylation state of a protein isolated in small quantities from a biological sample remains far f rom routine.