Characterization of Site-Specific Glycosylation in Influenza A Virus Hemagglutinin Produced by Spodoptera frugiperda Insect Cell Line

Influenza hemagglutinin is a surface glycoprotein related to virus invasion and host immune system response. Understanding site specific glycosylation of hemagglutinin will increase our knowledge about virus evolution and can improve the design and quality of vaccines. In our study, we used glycoproteomic analysis based on multienzyme digestion followed by LC tandem MS analysis to determine the glycosylation of Influenza hemagglutinin (H1/A/California/04/2009) using the following steps: PNGaseF treatment combined with trypsin or pepsin digestion was used to determine the glycosites and glycan occupancy. Three enzymes, trypsin, AspN, and pepsin, were used separately to generate suitable glycopeptides for online LC tandem MS analysis. The glycan structure of a given glycopeptide was determined by collision-induced dissociation MS/MS fragmentation, and the peptide backbone information was provided by collision-induced dissociation (CID)-MS3 fragmentation. With this approach, 100% sequence coverage of the hemagglutinin sample was obtained. Six glycosylation sites fitting the sequon N-X-S/T were successfully confirmed, and the glycan heterogeneity as well as the ratios of glycoforms were determined at each site.