Targeted N-Linked Glycosylation Analysis of H5N1 Influenza Hemagglutinin by Selective Sample Preparation and Liquid Chromatography/Tandem Mass Spectrometry

Using liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of deglycosylated and intact glycopeptides from tryptic digests of whole influenza virus, we determined that the six predicted N-linked glycosylation sites within the N-terminal ectodomain of hemagglutinin (HA) from three selected H5N1 strains are occupied. The use of selective sample preparation strategies, including solid-phase extraction (SPE) of glycopeptides via hydrazide capture chemistry as well as hydrophilic interaction liquid chromatography (HILIC), sufficiently reduced sample complexity to allow determination of occupied glycosylation sites. The specific amino acid sequence of the tryptic glycopeptides for the identified sites varied slightly among strains, but the overall locations of the occupied glycosylation sites were conserved in the protein sequence. We used this knowledge of glycosylation site occupation to examine the glycans attached to these occupied sites on HA for a reassortant H5N1 strain grown in embryonated chicken eggs. By applying mass spectrometry-based methodologies for examining glycosylation to the study of influenza virus proteins, we can better understand the effect that this post-translational modification has upon the virulence and antigenicity of emerging strains.