Glycoproteomics Analysis Reveals Differential Expression of Site-Specific Glycosylation in Human Milk Whey during Lactation

Protein N-glycosylation in human milk whey plays a substantial role in infant health during postnatal development. Changes in site-specific glycans in milk whey reflect the needs of infants under different circumstances. However, the conventional glycoproteomics analysis of milk whey cannot reveal the changes in site-specific glycans because the attached glycans are typically enzymatically removed from the glycoproteins prior to analysis. In this study, N-glycoproteomics analysis of milk whey was performed without removing the attached glycans, and 330 and 327 intact glycopeptides were identified in colostrum and mature milk whey, respectively. Label-free quantification of site-specific glycans was achieved by analyzing the identified intact glycopeptides, which revealed 9 significantly upregulated site-specific glycans on 6 glycosites and 11 significantly downregulated site-specific glycans on 8 glycosites. Some interesting change trends in N-glycans attached to specific glycosites in human milk whey were observed. Bisecting GlcNAc was found attached to 11 glycosites on 8 glycoproteins in colostrum and mature milk. The dynamic changes in site-specific glycans revealed in this study provide insights into the role of protein N-glycosylation during infant development.