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Comprehensive structural glycomic characterization of the glycocalyxes of cells and tissues

Nature Protocols volume 15, pages 2668–2704 (2020)Cite this article

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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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Fig. 1: Summary of the workflow for cell sample collection and membrane extraction.
Fig. 2: Summary of the workflow for comprehensive LC-MS/MS analysis of the cell membrane glycocalyx.
Fig. 3: Examples of tissues with cell surface glycocalyxes analyzed by LC-MS/MS.
Fig. 4: Examples of LC-MS profiles from the Caco-2 cell line.
Fig. 5: Glycoproteomic data of serum standard.
Fig. 6: Tandem MS spectra of selected compounds.
Fig. 7: Examples of site-specific glycopeptide mapping of N-linked glycoproteins and O-linked glycoproteins.
Fig. 8: The N-glycomic analysis of Caco-2 cells cultured grown at different days.
Fig. 9: The identification and quantitation of glycosphingolipids from Caco-2 and PNT2 cell lines.

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Article 23 June 2022

Ieva Bagdonaite, Stacy A. Malaker, … Nichollas E. Scott

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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Authors and Affiliations

  1. Department of Chemistry, University of California, Davis, Davis, California, USA

    Qiongyu Li, Yixuan Xie, Maurice Wong & Carlito B. Lebrilla

  2. Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, Davis, California, USA

    Mariana Barboza

  3. Department of Biochemistry and Molecular Medicine, University of California, Davis, Davis, California, USA

    Carlito B. Lebrilla

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Contributions

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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Correspondence to Carlito B. Lebrilla.

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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.

Reporting Summary

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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