Department of Glyco-Oncology and Medical Biochemistry, Osaka International Cancer Institute, 3-1-69 Otemae, Chuo-ku, Osaka 541-8567, Japan.
Center for Joint Research Facilities Support, Research Promotion and Support Headquarters, Fujita Health University, Toyoake, Aichi 470-1192, Japan.
STAR Protoc. 2021 Feb 3;2(1):100316. doi: 10.1016/j.xpro.2021.100316. eCollection 2021 Mar 19.
N-glycosylation is a fundamental post-translational protein modification in the endoplasmic reticulum of eukaryotic cells. The biosynthetic and catabolic flux of N-glycans in eukaryotic cells has long been analyzed by metabolic labeling using radiolabeled sugars. Here, we introduce a non-radiolabeling protocol for the isolation, structural determination, and quantification of N-glycan precursors, dolichol-linked oligosaccharides, and the related metabolites, including phosphorylated oligosaccharides and nucleotide sugars. Our protocol allows for capturing of the biosynthesis and degradation of N-glycan precursors at steady state. For complete details on the use and execution of this protocol, please refer to Harada et al. (2013), Harada et al. (2020), and Nakajima et al. (2013).
N-糖基化是真核细胞内质网中一种基本的翻译后蛋白质修饰。长期以来，人们一直通过使用放射性标记糖的代谢标记来分析真核细胞中 N-聚糖的生物合成和分解代谢通量。在这里，我们介绍了一种非放射性标记方案，用于分离、结构测定和定量 N-聚糖前体、多萜醇连接的寡糖以及相关代谢物，包括磷酸化寡糖和核苷酸糖。我们的方案允许在稳定状态下捕获 N-聚糖前体的生物合成和降解。有关此方案的使用和执行的完整详细信息，请参阅 Harada 等人（2013 年）、Harada 等人（2020 年）和 Nakajima 等人（2013 年）。
