细胞生物学
计算生物学
生物
凝集素
蛋白质组学
糖组
N-糖酰胺酶F
鉴定(生物学)
作者
Qiongyu Li,Yixuan Xie,Maurice Wong,Mariana Barboza,Carlito B. Lebrilla
出处
期刊:Nature Protocols
[Springer Nature]
日期:2020-07-17
卷期号:15 (8): 2668-2704
被引量:17
标识
DOI:10.1038/s41596-020-0350-4
摘要
The glycocalyx comprises glycosylated proteins and lipids and fcorms the outermost layer of cells. It is involved in fundamental inter- and intracellular processes, including non-self-cell and self-cell recognition, cell signaling, cellular structure maintenance, and immune protection. Characterization of the glycocalyx is thus essential to understanding cell physiology and elucidating its role in promoting health and disease. This protocol describes how to comprehensively characterize the glycocalyx N-glycans and O-glycans of glycoproteins, as well as intact glycolipids in parallel, using the same enriched membrane fraction. Profiling of the glycans and the glycolipids is performed using nanoflow liquid chromatography–mass spectrometry (nanoLC-MS). Sample preparation, quantitative LC–tandem MS (LC-MS/MS) analysis, and data processing methods are provided. In addition, we discuss glycoproteomic analysis that yields the site-specific glycosylation of membrane proteins. To reduce the amount of sample needed, N-glycan, O-glycan, and glycolipid analyses are performed on the same enriched fraction, whereas glycoproteomic analysis is performed on a separate enriched fraction. The sample preparation process takes 2–3 d, whereas the time spent on instrumental and data analyses could vary from 1 to 5 d for different sample sizes. This workflow is applicable to both cell and tissue samples. Systematic changes in the glycocalyx associated with specific glycoforms and glycoconjugates can be monitored with quantitation using this protocol. The ability to quantitate individual glycoforms and glycoconjugates will find utility in a broad range of fundamental and applied clinical studies, including glycan-based biomarker discovery and therapeutics. This protocol describes nanoflow liquid chromatography–mass spectrometry (nanoLC-MS) analysis of the N-glycans and O-glycans of glycoproteins and glycolipids, as well as site-specific glycosylation of membrane proteins.
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