![]() ![]() Protein glycosylation is a common post-translational modification process that governs a diverse range of biological functions involved in cell signaling, cell–cell contact, innate immune response, protein stability, and host–pathogen interactions. Viral fitness requires a balance of HA and NA functions to maintain the ability to infect and to release from host cells efficiently. Following replication, NA cleaves SA from glycans that release virions from host cells. HA binds to host cell sialylated glycan receptors and initiates viral infection. 1 The external parts of both HA and NA proteins consist of a stalk and globular head regions. Segments four and six encode two surface glycoproteins, the hemagglutinin (HA), a trimeric lectin, and the neuraminidase (NA), a tetrameric enzyme. Influenza A viruses belong to the Orthomyxoviridae family and contain a genome consisting of eight single-stranded, negative sense RNA segments. 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. Collisional cross section (CCS) provided by the ion mobility spectrometry from the timsTOF Pro MS data differentiated sialylation linkages of the glycopeptides. 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. 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). 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. 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. ![]() 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.
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