A Flexible Proteomic Approach to A2 Bovine Milk Authentication Through Tryptic, Thermolytic, and Peptic Proteotypic Peptides Elucidated by Routine LC–MS Analysis

The most common genotype for beta casein (β-CN) in European dairy cattle is A1A2, producing A1 and A2 variants merged in most traded bovine milks. A premium A2-milk category emerged worldwide through selection of A2A2-genotyped cows to produce added-value milk. Absence of A1 β-CN in bovine milk was assessed by a straightforward, cost-effective bottom-up low-resolution liquid chromatography-mass spectrometry (MS) methodology powered by ion-trap analysis. A preliminary data-dependent MS/MS exploratory approach characterized A1 and A2 variants in milk extracts digested with trypsin, pepsin, and thermolysin. Relative quantitation of selected peptide biomarkers was performed through Selected Reaction Monitoring analysis, detecting levels of 0.1% of A1 β-CN in milk mixtures from A1A1/A2A2-genotyped cows. Besides effective tryptic digestion, alternative thermolysin yielded short peptides that facilitated separation, while acidic pepsin supports the development of low-cost procedures. Affordability and robustness of this strategy favor its implementation by control laboratories addressing the authentication of A2 milk.