Laboratory of Bacterial Polysaccharides, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research , Food and Drug Administration , Silver Spring , Maryland 20993 , United States.
Genovis Inc. , Cambridge , Massachusetts 02142 , United States.
Anal Chem. 2018 Jul 3;90(13):8261-8269. doi: 10.1021/acs.analchem.8b01834. Epub 2018 Jun 24.
Glycosylation plays a critical role in the biosynthetic-secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Over 50% of mammalian cellular proteins are typically glycosylated; this modification is involved in a wide range of biological functions such as barrier formation against intestinal microbes and serves as signaling molecules for selectins and galectins in the innate immune system. N-linked glycosylation analysis has been greatly facilitated owing to a range of specific enzymes available for their release. However, system-wide analysis on O-linked glycosylation remains a challenge due to the lack of equivalent enzymes and the inherent structural heterogeneity of O-glycans. Although O-glycosidase can catalyze the removal of core 1 and core 3 O-linked disaccharides from glycoproteins, analysis of other types of O-glycans remains difficult, particularly when residing on glycopeptides. Here, we describe a novel chemoenzymatic approach driven by a newly available O-protease and solid phase platform. This method enables the assignment of O-glycosylated peptides, N-glycan profile, sialyl O-glycopeptides linkage, and mapping of heterogeneous O-glycosylation. For the first time, we can analyze intact O-glycopeptides generated by O-protease and enriched using a solid-phase platform. We establish the method on standard glycoproteins, confirming known O-glycosites with high accuracy and confidence, and reveal up to 8-fold more glycosites than previously reported with concomitant increased heterogeneity. This technique is further applied for analysis of Zika virus recombinant glycoproteins, revealing their dominant O-glycosites and setting a basis set of O-glycosylation tracts in these important viral antigens. Our approach can serve as a benchmark for the investigation of protein O-glycosylation in diseases and other biomedical contexts. This method should become an indispensable tool for investigations where O-glycosylation is central.
糖基化在内质网 (ER) 和高尔基体中的生物合成-分泌途径中起着关键作用。超过 50%的哺乳动物细胞蛋白通常被糖基化；这种修饰参与了广泛的生物学功能，例如形成对肠道微生物的屏障，并作为天然免疫系统中选择素和半乳糖凝集素的信号分子。由于有一系列可用的特定酶来释放它们，N-连接糖基化分析得到了极大的促进。然而，由于缺乏等效的酶和 O-聚糖固有的结构异质性，系统范围的 O-连接糖基化分析仍然是一个挑战。尽管 O-糖苷酶可以催化糖蛋白中核心 1 和核心 3 O-连接二糖的去除，但其他类型的 O-聚糖的分析仍然很困难，特别是当它们位于糖肽上时。在这里，我们描述了一种由新可用的 O-蛋白酶和固相平台驱动的新型化学酶方法。这种方法可以对 O-糖肽进行分配、N-聚糖谱分析、唾液酸化 O-糖肽连接分析以及异质 O-糖基化的映射。我们首次可以分析由 O-蛋白酶产生并使用固相平台富集的完整 O-糖肽。我们在标准糖蛋白上建立了该方法，高度准确和置信地确认了已知的 O-糖基化位点，并揭示了比以前报道的多达 8 倍的糖基化位点，同时增加了异质性。该技术进一步应用于寨卡病毒重组糖蛋白的分析，揭示了它们的主要 O-糖基化位点，并为这些重要病毒抗原中的 O-糖基化轨迹建立了一个基础集。我们的方法可以作为研究疾病和其他生物医学背景下蛋白质 O-糖基化的基准。这种方法应该成为 O-糖基化是中心的研究不可或缺的工具。
