Separation of phospho- and non-phosphopeptides using reverse phase column chromatography

Peptides containing phosphoserine, phosphothreonine or phosphotyrosine and their parent non-phosphorylated forms were chromatographed using standard C18 reverse phase chromatography in the presence of a water/acetonitrile gradient supplemented with different counter ions. We obtained the best separation of phosphorylated from non-phosphorylated Peptides in the presence of heptafluorobutyric acid, with differences in retention times as large as approximately 20 min. The chromatographic method was reliable in separation of the same Peptides phosphorylated at different positions, acidic or basic phospho-Ser/Thr-peptides or phospho-Tyr-containing Peptides. The described separation conditions are useful in studying the kinetics of phosphorylation/dephosphorylation and in analysis of phosphorylation sites in vivo.