Immobilized Enzyme Microreactors for Efficient Analysis of Tryptic Peptides in β-Casein and β-Lactoglobulin

Abstract In this study, our primary aim was to design and implement a rapid and effective analytical method for the examination of trypsin-digested peptides of two model proteins found in cow's milk: β -casein and β -lactoglobulin. These proteins were selected for their abundant nutrient content and functional attributes, notwithstanding their structural and compositional differences. We employed two methods for this purpose: the traditional in-gel protein digestion method and protein digestion using immobilized enzyme microreactors (μ-IMER). Both techniques made use of ZipTip pipette tips filled with C18 reverse phase media. These tips served to concentrate and purify the peptide samples, with the objective of determining the influence of such sample preparation on the improvement or reduction of sequence coverage results. In the preparation of the microreactor, we utilized a monolithic copolymer synthesized from GMA and EDMA. This was followed by surface modifications to attach the enzyme with the highest efficiency. We then evaluated the efficiency of the prepared microreactor under HPLC chromatographic conditions using a small-molecule trypsin substrate, BAEE. In the final stage, we analyzed the hydrolysates obtained from both the microreactor and the traditional digestion in solution method, with and without the presence of ZipTip pipette tips, using MALDI-TOF MS. This comprehensive approach facilitated a thorough study of peptides in β -casein and β -lactoglobulin, thereby enhancing our understanding of these vital cow's milk proteins.