Immobilized Fe3+ affinity chromatographic isolation of phosphopeptides

Immobilized Fe3+ affinity chromatography is suggested as a means of concentrating phosphopeptides that are present in too low a proportion in a complex mixture to be purified by a single-step method. A high-performance liquid chromatographic system and a chelating Superose HR 10/2 column were used. The chromatographic conditions were optimized using a tryptic hydrolysate of whole casein. The unbound fractions did not contain any phosphorylated peptide. All caseinophosphopeptides were retained. Only four other strongly basic peptides were also retained. The quantitative accuracy of the method was evaluated. This method allowed the isolation of phosphopeptides in gastric effluents of calves fed with milk.