Identification of N-linked glycoproteins in human milk by hydrophilic interaction liquid chromatography and mass spectrometry

Breastfeeding is now generally recognized as a critical factor in protecting newborns against infections. An important mechanism responsible for the antibacterial and antiviral effects of breast milk is the prevention of pathogen adhesion to host cell membranes mediated by a number of glycoconjugates, also including glycoproteins. A number of approaches to describe the complexity of human milk proteome have provided only a partial characterization of restricted classes of N-linked glycoproteins. To achieve this objective, profiling N-linked glycoproteins of human milk was performed by Hydrophilic Interaction LC (HILIC) and MS analysis. Glycopeptides were selectively enriched from the protein tryptic digest of human milk samples. Oligosaccharide-free peptides obtained by peptide N-glycosidase F (PNGase F) treatment were characterized by a shotgun MS-based approach, allowing the identification of N-glycosylated sites localized on proteins. Using this strategy, 32 different glycoproteins were identified and 63 N-glycosylated sites encrypted in them were located. The glycoproteins include immunocompetent factors, membrane fat globule-associated proteins, enzymes involved in lipid degradation and cell differentiation, specific receptors, and other gene products with still unknown functions.