New insights into site-specific N-glycosylation profiling differences of serum proteomes between colostrum and mature breast milk

N-glycosylation has important implications for the physicochemical properties and biochemical activity of breast milk proteins. However, an in-depth characterization of site-specific N-glycans in breast milk serum proteins is lacking. In this study, 875 site-specific N-glycans attached to 114 serum glycoproteins from breast colostrum (BC) and 427 site-specific N-glycans attached to 60 serum glycoproteins from breast mature milk (BM) were identified and quantified using label-free site-specific glycoproteomics. Among them, 24 site-specific N-glycans mapping to 11 N-glycosites on 9 glycoproteins were significantly increased, and 76 site-specific N-glycans mapping to 19 N-glycosites on 13 glycoproteins were significantly decreased. Gene ontology annotation analysis showed that BC and BM serum N-glycoproteins were mainly enriched in complement activation, classical pathway, extracellular exosome part, and immunoglobulin receptor binding. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that BC and BM serum N-glycoproteins were mainly involved in the PI3K-Akt signaling pathway. Additionally, breast milk serum N-glycoproteins carried fucosylated core structures as well as Lewis and sialylated branch structures that may contribute to neonatal retinal homeostasis. These results provide a molecular basis for understanding the unique biological activities of breast milk serum N-glycoproteins.