Targeted detection and quantification of casein macropeptide (variant A) by liquid chromatography-tandem mass spectrometry

Casein macropeptide (CMP) is an ideal ingredient for infant formula as well as special medical foods for patients suffering from phenylketonuria, but its large molecular mass and diversified glycosylation bring obstacles to its quantification in these products. Herein, we carried out enzymatic digestion simulation combined with database search and three proteotypic peptides with no glycosylation sites from CMP were screened out to represent CMPA isoform, one of the main variants of CMP, and avoid differences caused by glycosylation. The intensity of primary mass spectrometry, specificity (homology of peptide sequences), and degree of hydrolysis of three proteotypic peptides were evaluated, and target peptide 2 (147–152, TP2) was finally chosen as the quantitative peptide. A linear range of 2 ng/mL-2 μg/mL and a detection limit of 0.02 ng/mL for CMP was achieved by HPLC-MS/MS with isotope dilution. The recoveries of spiked TP2 in CMP hydrolysate were between 70% and 120%, and the strategy worked well in commercial formula samples.