Comparative N‑glycoproteomics Characterization of N‑glycoproteins of H3N8 Viruses Produced in Chicken Embryos versus MDCK Cells

N-linked glycosylation has been demonstrated to play a critical role in the folding and immunomodulation of influenza virus glycoproteins. Chicken embryos have historically been the predominant production host for influenza vaccines; however, Madin-Darby canine kidney (MDCK) cells have recently emerged as a significant alternative host. N-glycosylation of H3N8 viruses produced in chicken embryos and MDCK cells was comparatively characterized by mass spectrometry-based N-glycoproteomics with isotopic labeling at the molecular level of intact N-glycopeptides. A total of 3647 and 912 site- and structure-specific intact N-glycopeptides with glycosite localization and N-glycan monosaccharide sequence structure confirmation were identified from the whole N-glycoproteome and hemagglutinin N-glycoproteins, respectively, which correspond to 308 unique N-glycan sequence structures (defined by specific chemical linkages and branching patterns) and are derived from 171 distinct monosaccharide compositions. The data additionally contains N-glycosylation of the noncanonical glycosylation motif Asn-X-Cys. Ultimately, 628 intact N-glycopeptides were quantified as differentially expressed intact N-glycopeptides (DEGPs), where 425 were down-regulated and 203 were up-regulated. The results provide a global view of N-glycosylation of H3N8 viruses produced from the two systems, which has implications for virus research, including vaccine development, and the method could be extended to the N-glycosylation characterization of other viruses.N-连接糖基化（N-linked glycosylation）已被证实对流感病毒糖蛋白的折叠与免疫调节发挥关键调控作用。鸡胚历来是流感疫苗的主流生产宿主，但近年来Madin-Darby犬肾（Madin-Darby canine kidney, MDCK）细胞已成为重要的替代生产宿主。本研究采用基于质谱的N-糖基蛋白质组学技术，通过对完整N-糖肽进行同位素标记的分子层面分析，对比表征了鸡胚与MDCK细胞中培养的H3N8病毒的N-糖基化修饰模式。从全N-糖基蛋白质组与血凝素N-糖蛋白组分中，分别鉴定得到3647条与912条具备糖基化位点定位与N-聚糖单糖序列结构确证的位点特异性与结构特异性完整N-糖肽，这些糖肽对应308种独特的N-聚糖序列结构（以特定化学键连接方式与分支模式界定），并源自171种不同的单糖组成。本数据集还涵盖了非经典糖基化基序Asn-X-Cys的N-糖基化修饰信息。最终，共定量得到628条差异表达完整N-糖肽（differentially expressed intact N-glycopeptides, DEGPs），其中425条表达下调，203条表达上调。本研究结果为两种生产体系中制备的H3N8病毒的N-糖基化修饰提供了全局视角，其研究发现对包括疫苗开发在内的病毒研究具有参考价值，且该方法可推广至其他病毒的N-糖基化表征工作。