Sensitive and Specific Global Cell Surface N-Glycoproteomics Shows Profound Differences Between Glycosylation Sites and Subcellular Components

Cell surface glycans are essential for establishing cell communication, adhesion, and migration. However, it remains challenging to obtain cell surface-specific information about glycoconjugate structures. Acquiring this information is essential for unraveling the functional role of glycans and for exploiting them as clinical targets. To specifically analyze the N-glycoprotein forms expressed at the cell surface, we developed a C18 liquid chromatography (LC)-mass spectrometry (MS)-based glycoproteomics method in combination with highly specific cell surface protein labeling and enrichment using a biotin label. The surface-specificity of the method was validated by MS-based proteomics of subcellular component marker proteins. Using the human keratinocytes N/TERT-1 as a model system, we identified and quantified the glycosylation of hundreds of cell surface N-glycosylation sites. This approach allowed us to study the glycoforms present at the functional relevant cell surface, omitting immaturely glycosylated proteins present in the secretory pathway. Interestingly, the different stages of N-glycan processing at individual sites displayed at the cell surface were found to correlate with their accessibility for ER-residing processing enzymes, as investigated through molecular dynamics simulations. Using the new approach, we compared N-glycosylation sites of proteins expressed on the cell surface to their counterparts in a total cell lysate, showing profound differences in glycosylation between the subcellular components and indicating the relevance of the method for future studies in understanding contextual glycan functions.

细胞表面聚糖（cell surface glycans）对于维持细胞通讯、黏附与迁移过程至关重要。然而，获取针对细胞表面的糖缀合物（glycoconjugate）结构信息仍颇具挑战。获取此类信息对于阐明聚糖的功能机制，并将其开发为临床靶点具有关键意义。为特异性分析细胞表面表达的N-糖基化蛋白（N-glycoprotein）形式，本研究开发了一种基于C18液相色谱（LC）-质谱（MS）的糖蛋白质组学方法，并结合了采用生物素标记的高特异性细胞表面蛋白标记与富集策略。该方法的细胞表面特异性通过亚细胞组分标记蛋白的质谱蛋白质组学分析得到了验证。以人角质形成细胞N/TERT-1作为模型体系，本研究鉴定并定量了数百个细胞表面N-糖基化位点的糖基化修饰水平。该方法使我们能够研究功能相关的细胞表面存在的糖型，同时排除分泌通路中未成熟糖基化蛋白的干扰。有趣的是，通过分子动力学模拟分析发现，细胞表面各位点的N-聚糖加工不同阶段，与其对内质网（endoplasmic reticulum, ER）驻留加工酶的可及性存在显著相关性。利用该新方法，我们将细胞表面表达蛋白的N-糖基化位点与全细胞裂解液中的对应位点进行了比较，结果显示不同亚细胞组分的糖基化模式存在显著差异，这表明该方法对于未来研究语境依赖的聚糖功能具有重要应用价值。