Comparative N‑glycoproteomics Characterization of N‑glycoproteins of H3N8 Viruses Produced in Chicken Embryos versus MDCK Cells

N-linked glycosylation has been demonstrated to play
a critical
role in the folding and immunomodulation of influenza virus glycoproteins.
Chicken embryos have historically been the predominant production
host for influenza vaccines; however, Madin-Darby canine kidney (MDCK)
cells have recently emerged as a significant alternative host. N-glycosylation
of H3N8 viruses produced in chicken embryos and MDCK cells was comparatively
characterized by mass spectrometry-based N-glycoproteomics with isotopic
labeling at the molecular level of intact N-glycopeptides. A total
of 3647 and 912 site- and structure-specific intact N-glycopeptides
with glycosite localization and N-glycan monosaccharide sequence structure
confirmation were identified from the whole N-glycoproteome and hemagglutinin
N-glycoproteins, respectively, which correspond to 308 unique N-glycan
sequence structures (defined by specific chemical linkages and branching
patterns) and are derived from 171 distinct monosaccharide compositions.
The data additionally contains N-glycosylation of the noncanonical
glycosylation motif Asn-X-Cys. Ultimately, 628 intact N-glycopeptides
were quantified as differentially expressed intact N-glycopeptides
(DEGPs), where 425 were down-regulated and 203 were up-regulated.
The results provide a global view of N-glycosylation of H3N8 viruses
produced from the two systems, which has implications for virus research,
including vaccine development, and the method could be extended to
the N-glycosylation characterization of other viruses.