SEPARATION OF PHOSPHOPEPTIDES 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/a cetonitrile gradient supplemented with different counter ions. We obta ined the best separation of phosphorylated from non-phosphorylated pep tides in the presence of heptafluorobutyric acid, with differences in retention times as large as similar to 20 min. The chromatographic met hod was reliable in separation of the same peptides phosphorylated at different positions, acidic or basic phospho-Ser/Thr-peptides or phosp ho-Tyr-containing peptides. The described separation conditions are us eful in studying the kinetics of phosphorylation/ dephosphorylation an d in analysis of phosphorylation sites in vivo.