Imaged capillary isoelectric focusing and online mass spectrometry for milk whey protein characterization in dairy products

Characterizing major bovine milk proteins, including whey and casein, is of significant interest in the dairy industry. The diverse array of protein proteoforms can be different in terms of genetic variation, breed ways, lactation stage, and animal nutritional status. Current routine methods for bovine milk protein profiling are typically based on immunological techniques, infrared spectroscopy, slab gel isoelectric focusing, capillary electrophoresis, and high-performance liquid chromatography. However, there are obvious disadvantages of existing approaches including low throughput, tedious operation, unsatisfactory repeatability, and lack of robust quantitation capability. In this study, we present a novel approach that, for the first time, combines imaged capillary isoelectric focusing with mass spectrometry to separate and characterize whey proteins in milk products. The established method provided a rapid, repeatable, accurate, and simultaneous analysis of α-lactalbumin, β-lactoglobulin A, and β-lactoglobulin B within 10 min for diverse bovine milk samples. The methodology was systematically validated regarding repeatability of pI and peak area, sensitivity, linearity and recovery. The integration of high-resolution mass spectrometry with nano-electrospray ionization and icIEF has been pivotal in accurately identifying intact whey proteins in milk products. This approach has significantly enhanced the precise characterization of protein proteoforms in milk.