Comprehensive analysis of lactoferrin N-glycans with site-specificity from bovine colostrum using specific proteases and RP-UHPLC-MS/MS.

Bovine Lf (bLf) possesses five N-glycosylation sites having diverse glycans that alter over lactation and are
extensively characterised in milk. However, detailed site-specific characterisation in colostrum is limited.
In the present study, the glycan diversity in colostrum bLf from native and commercial preparations was
determined by generating N-glycopeptides using trypsin and a combination of trypsin/chymotrypsin
followed by RP-UHPLC-Q-TOF MS/MS. The glycan diversity at N300KS site is poorly understood and the
analysis revealed the presence of mostly biantennary complex glycans with core fucosylation and differential
sialylation. N252NS and N564DT sites were found to possess predominantly high mannose glycans.
A range of complex, hybrid and high mannose glycans were determined at N387VT and N495QT sites.
Glycan heterogeneity with relative abundance was determined on all glycosylation sites in bLf. In
addition, the glycan diversity of lipocalin-2 (N85VT) that might have co-purified in the commercial
colostrum bLf sample was also elucidated.