N-glycosylation of milk proteins: A review spanning 2010–2022

Nutrients in milk are abundant, and the rapid development of biomass spectrometry and bioinformatics algorithms has provided technical support to further elucidate nutrient content in milk such as N-glycoproteins, which are important post-translational modifications of proteins that have attracted significant research attention for their unique physicochemical and functional properties. We summarize the synthetic pathways of protein N-glycosylation, types of N-glycans, and primary distribution characteristics of N-glycoproteins in milk. Our review covers existing research of MS-based milk protein N-glycosylation in three dimensions: N-glycans, deglycosylated and intact N-glycopeptides obtained by enzymatic digestion of glycoproteins. A brief overview of protein N-glycosylation in dairy processing, absorption, and metabolism as well as biological functions is presented. The limitations and directions for future research on milk protein N-glycosylation are summarized. Intact N-glycopeptides obtained by enzymatic digestion of glycoproteins in current studies of milk protein N-glycosylation are insufficiently characterized and the construction of linkages between N-glycans and glycosylation sites must be further enhanced as a prerequisite for exploring milk protein N-glycosylation. N-glycosylation in dairy processing stabilizes protein conformation. Evidence from in vivo and in vitro studies suggests that milk protein N-glycans can be metabolised by intestinal microorganisms, modulate gut microbiota, reduce pathogen adhesion, regulate immune function, alleviate necrotizing enterocolitis, and affect brain development, with different structures having specific biological functions. However, more research is needed to validate existing findings. • The state of research on milk protein N-glycosylation is outlined. • The structure and composition of milk protein N-glycans are compared systematically. • The N-glycosites and site-specific N-glycosylation in milk are reviewed. • The importance of milk protein N-glycosylation is discussed. • Future research trends for milk protein N-glycosylation are presented.