Tryptic phosphopeptides from whole casein. I. Preparation and analysis by fast protein liquid chromatography

Abstract

Summary Tryptic phosphopeptides were obtained from whole bovine casein by chromatography on the anion exchange resin QAE-Sephadex A 25. Salt gradient elution of the column allowed separation of non-phosphorylated peptides from phosphorylated species. The preparations obtained contained at least seven distinct phosphopeptides of which the following casein fragments were identified: α s1 (43–58):2P, α s1 (59–79): 5P, α s2 (46–70): 4P, β(1–28): 4P, β(2–28): 4P, and β(33–48): 1P. Fast protein liquid chromatography (FPLC) on Mono Q HR 5/5 resin showed that the phosphopeptides were eluted in the same order as from the QAE-Sephadex resin. However, on the analytical column HR 5/5 the fragments α s1 (59–79): 5P and β(2–28): 4P, having the same net charge under the conditions of chromatography, co-eluted, whereas they were at least partly separated on the preparative column HR 16/10. Following enzymic dephosphorylation, the peptides eluted at lower salt strength in the gradient. FPLC on Mono Q resin thus permitted dephosphorylation to be monitored and intermediates between the parent species and the fully dephosphorylated peptide to be identified.