Characterization and comparison site-specific N-glycosylation profiling of milk fat globule membrane proteome in donkey and human colostrum and mature milk.

Milk fat globule membrane (MFGM) proteins are highly glycosylated and involved in various biological processes within the body. However, information on site-specific N-glycosylation of MFGM glycoproteins in donkey and human milk remains limited. This study aimed to map the most comprehensive site-specific N-glycosylation fingerprinting of donkey and human MFGM glycoproteins using a site-specific glycoproteomics strategy. We identified 1,360, 457, 2,617, and 986 site-specific N-glycans from 296, 77, 214, and 196 N-glycoproteins in donkey colostrum (DC), donkey mature milk (DM), human colostrum (HC), and human mature milk (HM), respectively. Bioinformatics was used to describe the structure-activity relationships of DC, DM, HC, and HM MFGM N-glycoproteins. The results revealed differences in the molecular composition of donkey and human MFGM N-glycoproteins and the dynamic changes to site-specific N-glycosylation of donkey and human MFGM glycoproteins during lactation, deepening our understanding of the composition of donkey and human MFGM N-glycoproteins and their potential physiological roles.

Quantitative label-free site-specific glycoproteomic analysis of the milk fat globule membrane protein in human colostrum and mature milk.

Human milk fat globule membrane (MFGM) proteins, which are N-glycosylated, play essential roles in neonatal development and physiological health. However, the profiles and landscape changes in the site-specific N-glycosylation of human MFGM proteins during lactation remain unclear. Therefore, in this study, based on an intact glycopeptide-centred strategy, 2617 unique site-specific N-glycans of 221 MFGM glycoproteins in human colostrum and 986 unique site-specific N-glycans of 200 MFGM glycoproteins in mature milk were characterised and quantified using label-free glycoproteomics. With milk maturation, 33 site-specific N-glycans on 10 N-glycoproteins increased significantly, and 113 site-specific N-glycans on 25 N-glycoproteins decreased significantly. Moreover, human MFGM glycoproteins with core-α1,6-fucosylated structures and Lewis and sialylated branching structures play a role in the biological processes of antigen processing and presentation. This study reveals the dynamic changes in human MFGM protein N-glycosylation patterns during lactation. Meanwhile, the study deepens our understanding of site-specific N-glycosylation of human MFGM glycoproteins. The results of the study provide a background reference for the development of infant formulas.