The coronavirus disease 2019 (COVID-19) pandemic has led to worldwide efforts to understand the biological traits of the newly identified human coronavirus (HCoV-19) virus. In this mass spectrometry (MS)-based study, we reveal that out of 21 possible glycosites in the HCoV-19 spike protein (S protein), 20 are completely occupied by N-glycans, predominantly of the oligomannose type. All seven glycosylation sites in human angiotensin I converting enzyme 2 (hACE2) were found to be completely occupied, mainly by complex N-glycans. However, glycosylation did not directly contribute to the binding affinity between HCoV-19 S protein and hACE2. Additional post-translational modification (PTM) was identified, including multiple methylated sites in both proteins and multiple sites with hydroxylproline in hACE2. Refined structural models of HCoV-19 S protein and hACE2 were built by adding N-glycan and PTMs to recently published cryogenic electron microscopy structures. The PTM and glycan maps of HCoV-19 S protein and hACE2 provide additional structural details for studying the mechanisms underlying host attachment and the immune response of HCoV-19, as well as knowledge for developing desperately needed remedies and vaccines.

2019 年冠状病毒病 (COVID-19) 大流行导致全世界努力了解新发现的人类冠状病毒 (HCoV-19) 病毒的生物学特征。在这项基于质谱 (MS) 的研究中，我们发现在 HCoV-19 刺突蛋白（S 蛋白）中的 21 个可能的糖位点中，有 20 个完全被N-聚糖占据，主要是寡甘露糖类型。发现人血管紧张素 I 转化酶 2 (hACE2) 中的所有七个糖基化位点都被完全占据，主要被复合物N-聚糖。然而，糖基化并没有直接促进 HCoV-19 S 蛋白和 hACE2 之间的结合亲和力。鉴定了额外的翻译后修饰 (PTM)，包括蛋白质中的多个甲基化位点和 hACE2 中具有羟脯氨酸的多个位点。通过在最近发表的低温电子显微镜结构中添加N-聚糖和 PTM，构建了 HCoV-19 S 蛋白和 hACE2 的精细结构模型。HCoV-19 S 蛋白和 hACE2 的 PTM 和聚糖图谱为研究宿主附着和 HCoV-19 免疫反应的潜在机制提供了额外的结构细节，以及开发急需的补救措施和疫苗的知识。