Abstract
Glycosphingolipids (GSLs) are ubiquitous glycoconjugates present on the cell membrane; they play significant roles in many bioprocesses such as cell adhesion, embryonic development, signal transduction and carcinogenesis. Analyzing such amphiphilic molecules is a major challenge in the field of glycosphingolipidomics. We provide a step-by-step protocol that uses a lectin microarray to analyze GSL glycans from cultured cells. The procedure describes (i) extraction of GSLs from cell pellets, (ii) N-monodeacylation using sphingolipid ceramide N-deacylase digestion to form lyso-GSLs, (iii) fluorescence labeling at the newly exposed amine group, (iv) preparation of a lectin microarray, (v) GSL-glycan analysis by a lectin microarray, (vi) complementary mass spectrometry analysis and (vii) data acquisition and analysis. This method is high-throughput, low cost and easy to conduct, and it provides detailed information about glycan linkages. This protocol takes ~10 d.
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Data availability
The authors declare that all the data generated or analyzed during this study are available within the protocol. Source data are provided with this paper.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (no. 81372365 and no. 81871955).
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H.D. and H.Y. designed and performed the experiments. H.D., H.Y. and F.Y. designed and performed the data acquisition and analysis. H.D. and Z.L. prepared the manuscript.
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Du, H. et al. Anal. Chem. 91, 10663–10671 (2019): https://doi.org/10.1021/acs.analchem.9b01945
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Du, H. et al. Anal. Chem. 91, 10663–10671 (2019): https://doi.org/10.1021/acs.analchem.9b01945
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Du, H., Yu, H., Yang, F. et al. Comprehensive analysis of glycosphingolipid glycans by lectin microarrays and MALDI-TOF mass spectrometry. Nat Protoc 16, 3470–3491 (2021). https://doi.org/10.1038/s41596-021-00544-y
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DOI: https://doi.org/10.1038/s41596-021-00544-y
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