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.