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Separation of phospho‐ and non‐phosphopeptides using reverse phase column chromatography
Author(s) -
Hiroshi Ohguro,
Krzysztof Palczewski
Publication year - 1995
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(95)00710-q
Subject(s) - phosphoserine , dephosphorylation , phosphopeptide , chromatography , chemistry , phosphorylation , reversed phase chromatography , peptide , high performance liquid chromatography , chromatographic separation , acetonitrile , phase (matter) , serine , biochemistry , phosphatase , organic chemistry
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 ∼ 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.