Abstract: Protein glycosylation is one of the most important post-translational modifications, which influences the functions of the modified proteins. The aim of this study was to elucidate the N-glycosylation of proteins in bovine milk. Using glycoproteomics, a total of 154 N-glycoproteins with 246 glycosylation sites were identified in bovine milk, including 117 N-glycoproteins with 183 glycosylation sites in colostrum, and 109 N-glycoproteins with 145 glycosylation sites in mature milk. The glycosylation site of N-283 on clusterin was expressed at the highest level in colostrum while N-93 on alphalactalbumin showed the highest expression in mature milk. Considering the quantitative and exclusive differences, 190 glycosylation sites on 129 glycoproteins were differentially expressed between colostrum and mature milk. According to gene ontology annotation, these differentially expressed glycoproteins participated in biological regulation, response to stimulus, multicellular organismal processes, localization and immune system processes; they were cellular components in the extracellular region and organelles and had various molecular functions such as binding, catalytic activity and molecular function regulators. The differentially expressed glycoproteins were mainly involved in the complement and coagulation cascades, Staphylococcus aureus infection and the lysosome pathways. In addition, some highly connected glycoproteins were identified through protein-protein interaction analysis. Our results enrich the knowledge on the glycoproteome of cow’s milk, including the N-glycoprotein composition and glycosylation sites and also reveal the functions of protein N-glycosylation in bovine milk whey, which provide a theoretical foundation for the evaluation and improvement of bovine milk quality, the development of infant formula closer to human milk, and the production of functional foods.

Key words: bovine colostrum, mature bovine milk, whey, N-glycoproteins, function analysis