Enhanced capillary electrophoresis-mass spectrometry with deactivated fused-silica capillaries for bovine milk protein profiling and A2 milk authentication

Profiling intact bovine milk proteins, including casein and whey proteins, is fundamental in nutrition and food technology. Routine analysis is typically performed using capillary electrophoresis with ultraviolet detection (CE-UV), a well-established technique that, however, does not allow unequivocal identification of the separated proteins. To overcome this limitation, we previously developed a capillary electrophoresis-mass spectrometry (CE-MS) method that enabled both separation and confident identification of major bovine milk proteins. This approach, nevertheless, relied on the preparation of lab-made hydroxypropyl cellulose (HPC)-coated capillaries to prevent protein adsorption on the capillary walls, a procedure that is laborious, time-consuming, and suffers from limited reproducibility between batches unless tightly controlled. In this study, we present for the first time a CE-MS method employing commercially available OV-1701-OH deactivated fused-silica capillaries. This advancement streamlines the workflow and improves repeatability, peak resolution, and capillary lifetime. The enhanced CE-MS method, together with the routine CE-UV approach, was applied to the analysis of milk samples from cows carrying the β-casein (β-CN) A1A1, A1A2, and A2A2 genotypes, as well as to quantify low-level contamination of A2A2 milk with A1A1 milk. Both methods demonstrated strong potential for comprehensive profiling of bovine casein and whey proteins, providing rapid and reliable tools for the authentication of the highly valued A2A2 milk, commonly referred to as A2. Importantly, accurate mass high-resolution MS proved essential for the precise characterization of β-CN A1 and A2 proteoforms, enabling unambiguous identification of milk based on its specific β-CN composition.