title: An unbiased approach for analysis of protein glycosylation and application to influenza vaccine hemagglutinin.
abstract: Here a mass spectrometry-based platform for the analysis of glycoproteins is presented. Glycopeptides and released glycans are analyzed, the former by quadrupole orthogonal time-of-flight liquid chromatography/mass spectrometry (QoTOF LC/MS) and the latter by permethylation analysis using matrix-assisted laser desorption/ionization (MALDI)-TOF MS. QoTOF LC/MS analysis reveals the stochastic distribution of glycoforms at occupied sequons, and the latter provides a semiquantitative assessment of overall protein glycosylation. Hydrophilic interaction chromatography (HILIC) was used for unbiased enrichment of glycopeptides and was validated using five model N-glycoproteins bearing a wide array of glycans, including high-mannose, complex, and hybrid subtypes such as sulfo and sialyl forms. Sialyl and especially sulfated glycans are difficult to analyze because these substitutions are labile. The conditions used here allow detection of these compounds quantitatively, intact, and in the context of overall glycosylation. As a test case, we analyzed influenza B/Malaysia/2506/2004 hemagglutinin, a component of the 2006-2007 influenza vaccine. It bears 11 glycosylation sites. Approximately 90% of its glycans are high mannose, and 10% are present as complex and hybrid types, including those with sulfate. The stochastic distribution of glycoforms at glycosylation sites is revealed. This platform should have wide applications to glycoproteins in basic sciences and industry because no apparent bias for any glycoforms is observed.
authors.affiliation: Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA.
authors.forename: Yanming
authors.initials: Y
authors.lastname: An
authors.forename: John F
authors.initials: JF
authors.lastname: Cipollo
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D054794:Glycomics / Q000379:methods; D006020:Glycopeptides / Q000032:analysis; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D006388:Hemagglutinins / Q000032:analysis*; D007252:Influenza Vaccines / Q000032:analysis*; D013058:Mass Spectrometry / Q000379:methods; D008958:Models, Molecular; D011134:Polysaccharides / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D006020:Glycopeptides; D006023:Glycoproteins; D006388:Hemagglutinins; D007252:Influenza Vaccines; D011134:Polysaccharides