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J Biomol Tech. 2010 September; 21(3 Suppl): S47.
PMCID: PMC2918135

Investigation of Host Cell Induced Variations in N-linked Glycosylation of Influenza Hemagglutinin by Liquid Chromatography-Tandem Mass Spectrometry

T. Williams, J. Pirkle, and J. Barr
Centers for Disease Control and Prevention, Atlanta, GA, United States



Influenza hemagglutinin (HA) is the primary component of seasonal/pandemic vaccines. HA N-linked glycosylation is thought to affect the virulence of an influenza strain by interfering with the cell recognition binding site or by masking antigenic regions of the protein. Identification of host cell imparted differences in the number/conservation of glycosylation sites as well as the size/complexity of the glycans themselves is necessary for determining the efficacy of alternative virus propagation platforms for vaccine production and is essential for pandemic preparedness. Whole virus samples from a single H5N1 strain (A/Vietnam/1203/2004) were grown in four different host cell systems: i) embryonated chicken eggs; ii) Madin-Darby canine kidney cells (MDCK); iii) Vero E6 cells; and iv) A549 human alveolar epithelial carcinoma cells. Prior to glycosylation analysis, total HA was quantified using liquid chromatography tandem mass spectrometry (LC-MS/MS). Hydrazide solid-phase extraction (SPE) was utilized to specifically isolate HA glycopeptides in order to determine site occupation via deglycosylation and subsequent LC-MS/MS. Hydrophilic interaction liquid chromatography (HILIC) was utilized to improve LC-MS/MS analysis of intact tryptic glycopeptides. De novo peptide sequencing was used for peptide confirmation. ExPASy's GlycoMod Tool was utilized to aid interpretation of intact glycopeptide MS/MS data. We report application of an approach for determining N-linked glycosylation site occupation and investigating the glycans attached to those sites for tryptic digests of whole influenza virus samples via selective sample preconcentration and LC-MS/MS analysis. We have used this data on glycosylation site occupation/conservation to assist in the examination of intact glycopeptides generated from tryptic digests of a single H5N1 stain grown in four different propagation systems. We have proposed potential sugar compositions for multiple glycoforms associated with the occupied glycosylation sites on HA. The information gained from these experiments may potentially be useful for examining the relationship between changes in HA glycosylation and changes in virulence/antigenicity.

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