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In this report, we have systematically evaluated the glycan composition, structural distribution and topology of glycosylation for two high-yield candidate reassortant vaccines (NIBRG-121xp and NYMC-X181A) by combining various enzymatic digestions with high performance liquid chromatography and multiple-stage mass spectrometry. Proteomic data analyses of the full-length protein sequences determined 9 N-glycosylation sites of hemagglutinin, and defined 6 N-glycosylation sites and the glycan structures of low abundance neuraminidase, which were occupied by high-mannose, hybrid and complex-type N-glycans. A total of ~300 glycopeptides were analyzed and manually validated by tandem mass spectrometry. The specific N-glycan structure and topological location of these N-glycans are highly correlated to the spatial protein structure and the residential ligand binding. Interestingly, sulfation, fucosylation and bisecting N-acetylglucosamine of N-glycans were also reliably identified at the specific glycosylation sites of the two influenza proteins that may serve a crucial role in regulating the protein structure and increasing the protein abundance of the influenza virus reassortants.<\/jats:p>","DOI":"10.1038\/s41598-017-10714-2","type":"journal-article","created":{"date-parts":[[2017,8,25]],"date-time":"2017-08-25T11:59:59Z","timestamp":1503662399000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":38,"title":["Topological N-glycosylation and site-specific N-glycan sulfation of influenza proteins in the highly expressed H1N1 candidate vaccines"],"prefix":"10.1038","volume":"7","author":[{"given":"Yi-Min","family":"She","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aaron","family":"Farnsworth","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xuguang","family":"Li","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Terry D.","family":"Cyr","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2017,8,31]]},"reference":[{"key":"10714_CR1","doi-asserted-by":"publisher","first-page":"1836","DOI":"10.1016\/j.vaccine.2010.12.044","volume":"29","author":"JS Robertson","year":"2011","unstructured":"Robertson, J. 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