Caseinophosphopeptide enrichment and identification

Caseinophosphopeptides (CPPs) were generated via tryptic hydrolysis of sodium caseinate followed by heat treatment (75°C) at pH 6.0, 7.0 and 8.0. Calcium aggregation, ethanol precipitation and gallium immobilised metal affinity chromatography (IMAC) were applied to enrich the CPPs. Nano-liquid chromatography electrospray ionisation-quadrupole time-of-flight tandem mass spectrometry was used for phosphopeptide separation and identification. The combination of gallium IMAC isolation with calcium and ethanol precipitation appears to be a useful tool in characterising the fate of CPPs in CN hydrolysates during thermal processing. CPPs appeared to be less susceptible to thermal modification when heat-treated at pH 7.0 than at pH 6.0 and 8.0. Different extents of methionine oxidation occurred in the CPPs depending on heat-treatment pH. The results herein may have important implications for the manufacture of CPPs and for the quality control of CPP products.