Selection of marker peptides from casein phosphopeptide and application for quantification in infant formula

Casein phosphopeptides (CPPs) have been used worldwide as a nutritional supplement. However, the peptide components have been unknown; as a consequence, few quantification methods of CPP in infant formula were reported. This study introduced a quantification method based on peptide marker and corresponding peptide selection strategy using a simplified model with four commercial types of CPP. The peptides from four commercial CPPs were first identified. Due to the great variety of CPPs, two marker selection strategies were adopted: on one hand, universal marker peptide VLPVPQK can be used for the quantification of all four commercial CPPs, if the CPP can be obtained as a standard. On the other hand, the specific marker peptide LYQEPVLGPV can be used for identification and quantification of commercial CPP type K content in infant formula with a fixed calculation factor. In the simplified model, the combination use of the two markers can meet most of the requirements of CPP analysis in infant formula. The method validation revealed that this was suitable for the routine analysis laboratories without proteomics backgrounds. This selection strategy was suggested for the large-scale marker peptide selection with all commercial CPPs, which can give a comprehensive solution of CPP quantification in infant formula. Abbreviations: CPP: Casein phosphopeptides; LC: Liquid chromatography; TQMS: Triple quadrupole mass spectrometry; MRM: Multiple reaction monitoring; RSD: Relative standard deviation; L*: [¹³C₆, ¹⁵N]-leucine; SSSEE: Peptides sequence of serine-serine-serine-glutamic acid-glutamic acid.