Characterisation and comparison site-specific N-glycosylation of whey proteins from donkey and human colostrum and mature milk using glycoproteomics

Although the composition of donkey milk is similar to that of human milk, systematic comparisons of the sitespecific N-glycosylation patterns of their whey proteins are lacking. In this study, hydrophilic interaction chromatography-based enrichment of intact N-glycopeptides, coupled with a site-specific glycoproteomics strategy, was used to systematically characterise whey N-glycoproteins in donkey colostrum (DC), donkey mature milk (DM), human colostrum (HC), and human mature milk (HM) for the first time. We identified 628, 347, 868, and 425 site-specific N-glycans mapped to 135, 67, 113, and 60 glycoproteins in DC, DM, HC, and HM, respectively. Bioinformatic analysis revealed the potential biological effects of N-glycosylation modifications on the whey proteins themselves. Our findings elucidated the composition of donkey and human milk whey Nglycoproteins and their potential structure-activity relationships and provided guidance for the production of specific functional donkey milk products and the development of donkey milk-based infant formulas.