Glycoproteomics reveal differences in site-specific N-glycosylation of whey proteins between donkey colostrum and mature milk

Donkey whey protein has high nutritional value, yet systematic characterisation of site-specific N-glycosylation of donkey whey protein is still in its infancy. This study used label-free site-specific glycoproteomics to identify and quantify 685 intact N-glycopeptides from 135 whey glycoproteins in donkey colostrum (DC) and 384 intact N-glycopeptides from 67 glycoproteins in donkey mature milk (DM). As lactation progressed, 23 site-specific N-glycans mapping to 13 glycosites on 12 glycoproteins were significantly decreased, and 19 site-specific N-glycans mapping to 7 glycosites on 6 glycoproteins were significantly increased. We also found that certain N-glycans attached to the glycosites exhibited significant site specificity. This study provides new insights into the potential relationship between the biological function of donkey whey proteins and their N-glycosylation modifications. Furthermore, the study provides molecular evidence for differences in the site-specific N-glycosylation profile of donkey whey protein during lactation.