Identification of post-translational modifications of milk and whey proteins with different structures

Abstract

Post-translational modifications (PTMs) regulate cellular processes, and any disruption of PTMs leads to abnormal activity of biological processes, and therefore diseases. The main goal of the present research was focused on developing a rapid analytical method for identifying PTMs in milk (β-casein) and whey (β-lactoglobulin) proteins that differ by structure and composition; the chosen proteins are considered the richest source of nutrients and functional components. The classical in-gel protein digestion method and protein digestion in a microreactor (μ-IMER) method has been performed. In addition, ZipTip pipette tipscontaining C18 reverse phase media were used for both methods to concentrate and purify peptide samples; they also aimed to determine the effect of such a prepared sample on the improvement or deterioration of the sequence coverage result. As support for the preparation of the microreactor, a monolithic copolymer synthesized from GMA and EDMA was used. Subsequently, surface modifications were carried out to attach the enzyme with the highest efficiency. The efficiency of the prepared microreactor was evaluated under HPLC chromatographic conditions using a small-molecule trypsin substrate (BAEE). The obtained hydrolysates from the microreactor and the classical digestion in solution method in the presence of ZipTip pipette tips and without were analyzed by MALDI-TOF MS.