Separation and characterization of bovine milk proteins by capillary electrophoresis‐mass spectrometry

Protein profiling of major bovine milk proteins (i.e., whey and casein proteins) is of great interest in food science and technology. This complex set of protein proteoforms may vary with breed, genetics, lactation stage, health, and nutritional status of the animal. Current routine methods for bovine milk protein profiling at the intact level are typically based on capillary electrophoresis‐ultraviolet, which does not allow confirming unequivocally the identity of the separated proteins. As an alternative, in this study, we describe for the first time a novel and simple capillary electrophoresis‐mass spectrometry method in positive electrospray ionization mode. Under the optimized conditions, capillary electrophoresis‐mass spectrometry allowed the separation and identification at the intact level of major bovine milk whey and casein proteins in less than 15 min. Furthermore, high‐resolution mass spectrometry confirmed its importance in the reliable characterization of bovine milk protein proteoforms, especially those with slight molecular mass differences, such as β‐casein A1 and A2, which are relevant to unequivocally identify milk with specific β‐casein compositions (e.g., A2A2 milk, which is widely known as A2 milk). This differentiation was not possible by matrix‐assisted laser desorption/ionization mass spectrometry, which provided rapidly and easily a rich but less accurate fingerprint of bovine milk proteins due to the lower mass resolution.