Abstract
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.
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Data availability
The data are all available online. Data from ref. 23 (Park, D. et al. Glycobiology 27, 847–860 (2017)) (used for Fig. 4a) are available at https://doi.org/10.1093/glycob/cwx041. Data from ref. 24 (Park, D. et al. Chem. Sci. 9, 6271–6285 (2018)) (used for Figs. 4b and 9) are available at https://doi.org/10.1039/c8sc01875h. Data from ref. 26 (Wong, M. et al. Sci. Rep. 8, 10993 (2018)) (used for Fig. 4c) are available at https://doi.org/10.1038/s41598-018-29324-7. Data from ref. 25 (Li, Q. et al. Chem. Sci. 10, 6199–6209 (2019)) (used for Fig. 6) are available at https://doi.org/10.1039/c9sc01360a. Data from ref. 33 (Park, D. et al. Mol. Cell. Proteomics 14, 2910–2921 (2015)) (used for Fig. 8) are available at https://doi.org/10.1074/mcp.M115.053983.
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Acknowledgements
This work was supported by the National Institutes of Health (GMRO1R01, GM049077).
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Q.L., Y.X., M.W., M.B., and C.B.L. contributed to the development of this protocol and wrote and edited the manuscript.
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Key references using this protocol
Park, D. D. et al. Chem. Sci. 9, 6271–6285 (2018): https://doi.org/10.1039/c8sc01875h
Wong, M., Xu, G., Park, D., Barboza, M. & Lebrilla, C. B. Sci. Rep. 8, 10993 (2018): https://doi.org/10.1038/s41598-018-29324-7
Li, Q., Xie, Y., Xu, G. & Lebrilla, C. B. Chem. Sci. 10, 6199–6209 (2019): https://doi.org/10.1039/c9sc01360a
Supplementary information
Supplementary Information
Supplementary Figs 1 and 2.
Supplementary Data 1
The table contains examples of N-glycans with their masses, compositions, and types.
Supplementary Data 2
The table contains possible ceramide m/z values.
Supplementary Data 3
The table can be used to match the closest glycan composition to the glycosphingolipid with a known precursor m/z, precursor charge state, and ceramide m/z.
Supplementary Data 4
The table contains some common saccharide and lipid fragments.
Supplementary Data 5
The table contains a pivot table for generating a database of glycosphingolipids.
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Li, Q., Xie, Y., Wong, M. et al. Comprehensive structural glycomic characterization of the glycocalyxes of cells and tissues. Nat Protoc 15, 2668–2704 (2020). https://doi.org/10.1038/s41596-020-0350-4
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DOI: https://doi.org/10.1038/s41596-020-0350-4
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