Online trypsin digestion coupled with LC-MS/MS for detecting of A1 and A2 types of β-casein proteins in pasteurized milk using biomarker peptides.

Bovine A1-or A2-type β-caseins have attracted a growing interest due to their variation in beta-casomorphin-7 (BCM-7) formation, which may affect health. In the present work, identification and quantification of A1 and A2 types of β-casein proteins at the peptide level was achieved for the first time. An automated and online immobilized trypsin digestion system was employed for high throughput digesting of proteins into peptides. Tryptic peptides were separated and analyzed subsequently by liquid chromatography coupled to mass spectrometry platform. Two specific peptides ranging from the position of 49 to 97 in the peptide chain were selected for the identification and quantification of A1 and A2 β-casein, which covered the different amino acids between them. Synthetic isotopically labeled winged peptides were used for absolute quantification. Compared with traditional in-solution digestion, online digestion shortens digestion times from 2 to 24 h to 4 min. The limits of quantification (LOQ) of A1 and A2 β-casein in pasteurized milk are 0.8 and 2.4 µg/g, respectively. To further demonstrate the applicability of the proposed method, commercial pasteurized milk tests were performed with satisfactory results. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05376-6.