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.
Ohguro
H
H
Department of Ophthalmology, School of Medicine, University of Washington, Seattle 98195, USA.
Palczewski
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D051379:Mice; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D015203:Reproducibility of Results; D012243:Rhodopsin / Q000302:isolation & purification / Q000378:metabolism
D005136:Eye Proteins; D010748:Phosphopeptides; D012243:Rhodopsin; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; C496762:Grk1 protein, mouse