About 60% of the fucosylated N-glycans in human milk were reported to be multi-fucosylated, whereas this number was just 30% in bovine milk. Moreover, human milk exhibited more glycoforms harboring multiple fucoses when compared to bovine milk. They reported that 75% of the N-glycans in human milk correspond to fucosylated glycans, while this number was only 31% in bovine milk. (15) compared human and bovine milk and found substantial differences in their fucosylated N-glycans. Released N-glycans have revealed that a specific feature of human milk glycosylation is the high degree of abundant fucosylation, when compared with bovine milk (15) or healthy human serum. (14) used hydrophilic-interaction chromatography (HILIC) glycopeptide enrichment and applied this to a milk sample collected on day 7 after parturition, also merely analyzing the deglycosylated peptides, identifying 63 N-glycosylated sites originating from 32 glycoproteins. (12) investigated human colostrum and mature milk samples, and reported 68 and 58 N-glycoproteins and 111 and 96 N-glycosites, respectively, using a lectin-enrichment approach and subsequent identification of the deglycosylated peptides. (15,16) To name a few, a recent study from Cao et al. Recent studies have mainly focused on the mapping of N-glycosylation sites of enzymatic deglycosylated peptides (12−14) or on enzymatically released N-glycans.
Thus, further developing this analysis is essential for obtaining information on site-specific glycosylation. The glycopeptide-centric analysis of human milk protein glycosylation is still rather in its infancy.
(8) To understand the bioactive effects of the continuing changes of glycosylation in human milk, it is necessary that protein glycosylation analysis is done both in a personalized manner and longitudinally. (7) Like other components in human milk, protein glycosylation is quite diverse among individual mothers, and it is thought to also vary quite dynamically across lactation. For instance, glycosylated proteins are relevant to proteolytic susceptibility, (4) as competitive inhibitors of pathogen binding (5,6) and immunomodulators, (5) all together working to protect the infant’s health. (3) Protein glycosylation is of special interest in milk due to its widespread functional capacity.
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(1) Glycans form a major component of human milk, occurring both in free form as lactose and oligosaccharides, (2) as well as conjugated to glycoproteins primarily through N- and/or O-linked glycosylation. Human milk is a remarkable multifunctional fluid of the mammary gland, providing varying amounts of nutritional and non-nutritional, bioactive components to satisfy the specific demand of a newborn. Building on the improved glycoproteomics approach outlined above, future studies are warranted to reveal the potential impact of the observed glycosylation microheterogeneity on the healthy development of infants. For instance, fucosylation, especially terminal fucosylation, increased across the lactation period. Although each glycoprotein, N-glycosylation site, and attached glycan revealed distinct dynamic changes, we did observe a few general trends. We next quantified 287 of these glycopeptides originating from 50 glycoproteins using a targeted proteomics approach. We were able to map around 1700 glycopeptides from 110 glycoproteins covering 191 glycosites, of which 43 sites have not been previously reported with experimental evidence. Here, we describe an automated platform using hydrophilic-interaction chromatography (HILIC)-based cartridges enabling the proteome-wide monitoring of intact N-glycopeptides using just a digest of 150 μg of breast milk protein.
Due to the individual uniqueness of each mother’s milk and the overall complexity and temporal changes of protein N-glycosylation, analysis of the human milk N-glycoproteome requires longitudinal personalized approaches, providing protein- and N-site-specific quantitative information. Protein N-glycosylation on human milk proteins assists in protecting an infant’s health and functions among others as competitive inhibitors of pathogen binding and immunomodulators.
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