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Comprehensive analysis of glycosphingolipid glycans by lectin microarrays and MALDI-TOF mass spectrometry

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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Fig. 1: Flowchart illustrating the steps involved in this protocol.
Fig. 2: Lectin microarray analysis of GSL glycans from HL-7702, HMCC97L, HMCC97H and HCCLM3 cell lines.
Fig. 3: MALDI-TOF mass spectra of GSL glycans identified and annotated with proposed structures according to the results of lectin microarrays.
Fig. 4: MALDI-TOF/TOF-MS analysis of GSL-glycan precursor ions in the mass spectra.

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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Authors

Contributions

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.

Corresponding author

Correspondence to Zheng Li.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Ruijun Tian and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key reference using this protocol

Du, H. et al. Anal. Chem. 91, 10663–10671 (2019): https://doi.org/10.1021/acs.analchem.9b01945

Key data used in this protocol

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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