Direct nanoHPLC-ESI-QTOF MS/MS analysis of tryptic caseinophosphopeptides

Caseinophosphopeptides (CPPs) were generated following tryptic hydrolysis of sodium caseinate. Hydrolysate peptides were separated and identified using nano-HPLC ESI-QTOF MS/MS. Sequence coverage in the 3 h hydrolysate was 79.4%, 55.6%, 80.9% and 68.1% for αs1-, αs2-, β- and κ-casein (CN), respectively. Variable levels of serine phosphorylation in β-CN f1–25 were observed in the 3 h hydrolysate. Analysis of β-CN f1–25 4P demonstrated that this peptide was stable during the course of hydrolysis. The effect of heat treatment (75 °C, 45 min) at pH 6.0, 7.0 and 8.0 on the peptide profile of the 3 h hydrolysate was studied. Compared to pH 6.0 and 8.0, least modification in phosphopeptide profiles was observed for the hydrolysate sample heated at pH 7.0. Different dephosphorylation and oxidation patterns were also observed following heat treatment at the three pH values. These results demonstrate that heat treatment, in addition to pH, has a major effect on both the phosphorylated and non-phosphorylated peptide profiles of CN hydrolysates.