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.