Peptide Mapping and Purification of Phosphopeptides Using High-Performance Liquid Chromatography

This chapter describes peptide mapping and purification of phosphopeptides using high-performance liquid chromatography (HPLC). Protein phosphorylation is now firmly established as an important mechanism for regulation of diverse cellular functions, such as metabolism, membrane transport, muscle contraction, protein synthesis, neuronal activity, and cellular transformation by viruses. In addition, many phosphoproteins have multiple phosphorylation sites. One of the most complicated of these is skeletal muscle glycogen synthase, which contains at least seven phosphorylation sites per subunit. These multiple phosphorylations can be catalyzed by a number of different protein kinases. To establish the specificities of the kinases for the several phosphorylation sites, the HPLC methodology has been developed; this methodology is rapid and generally gives good recoveries of phosphopeptides.