Glycoproteomic Analyses of Influenza A Viruses Using timsTOF Pro MS

N-Linked glycosylation in hemagglutinin
and neuraminidase
glycoproteins of influenza viruses affects antigenic and receptor
binding properties, and precise analyses of site-specific glycoforms
in these proteins are critical in understanding the antigenic and
immunogenic properties of influenza viruses. In this study, we developed
a glycoproteomic approach by using a timsTOF Pro mass spectrometer
(MS) to determine the abundance and heterogeneity of site-specific
glycosylation for influenza glycoproteins. Compared with a Q Exactive
HF MS, the timsTOF Pro MS method without the hydrophilic interaction
liquid chromatography column enrichment achieved similar glycopeptide
coverage and quantities but was more effective in identifying low-abundance
glycopeptides. We quantified the distributions of intact site-specific
glycopeptides in hemagglutinin of A/chicken/Wuxi/0405005/2013 (H7N9)
and A/mute swan/Rhode Island/A00325125/2008 (H7N3). Results showed
that hemagglutinin for both viruses had complex N-glycans at N22, N38, N240, and N483 but only high-mannose glycans
at N411 and, however, that the type and quantities of glycans were
distinct between these viruses. Collisional cross section (CCS) provided
by the ion mobility spectrometry from the timsTOF Pro MS data differentiated
sialylation linkages of the glycopeptides. In summary, timsTOF Pro
MS method can quantify intact site-specific glycans for influenza
glycoproteins without enrichment and thus facilitate influenza vaccine
development and production.