Glycoproteomics analysis reveals differential site-specific N-glycosylation of donkey milk fat globule membrane protein during lactation

N-glycosylation is a prevalent and complex post-translational modification of milk proteins with significant biological importance. However, the systematic characterisation of donkey milk fat globule membrane (MFGM) N-glycoproteins remains largely ill-defined. Here, 1443 intact N-glycopeptides from 336 MFGM glycoproteins in donkey colostrum (DC) and 489 intact N-glycopeptides from 86 MFGM glycoproteins in donkey mature milk (DM) were identified via label-free site-specific glycoproteomics. Mannosylation and fucosylation were predominant in DC MFGM N-glycoproteins compared to sialylation and mannosylation in DM. Among them, 22 site-specific N-glycans attached to 14 glycosites of eight glycoproteins were significantly increased, whereas 30 site-specific N-glycans attached to 19 glycosites of 16 glycoproteins were significantly decreased. Furthermore, the site-specific N-glycans with Neu5Gc moieties or simultaneous fucosylation and sialylation were not significantly increased, exhibiting significant site specificity. We provide new insights into the composition of donkey MFGM N-glycoproteins and their roles in donkey milk-related biological functions.