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Shotgun glycomics: a microarray strategy for functional glycomics

Nature Methods volume 8, pages 85–90 (2011)Cite this article

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Abstract

Major challenges of glycomics are to characterize a glycome and identify functional glycans as ligands for glycan-binding proteins (GBPs). To address these issues we developed a general strategy termed shotgun glycomics. We focus on glycosphingolipids (GSLs), a class of glycoconjugates that is challenging to study, recognized by toxins, antibodies and GBPs. We derivatized GSLs extracted from cells with a heterobifunctional fluorescent tag suitable for covalent immobilization. We separated fluorescent GSLs by multidimensional chromatography, quantified them and coupled them to glass slides to create GSL shotgun microarrays. Then we interrogated the microarrays with cholera toxin, antibodies and sera from individuals with Lyme disease to identify biologically relevant GSLs that we subsequently characterized by mass spectrometry. Shotgun glycomics incorporating GSLs and potentially glycoprotein-derived glycans is an approach for accessing the complex glycomes of animal cells and is a strategy for focusing structural analyses on functionally important glycans.

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Figure 1: Schematic for shotgun glycomics.
Figure 2: Fluorescent derivatization of GSLs for shotgun glycomics.
Figure 3: Binding assay on the BBG–GSL-AOAB microarray prepared from two-dimensional HPLC separation.
Figure 4: Binding of sera from individuals with Lyme disease and control sera on the BBG microarray.
Figure 5: The GSL microarray from human erythrocytes and its interrogation with lectins and antibodies.

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Acknowledgements

This work was supported in part by a Bridge grant to R.D.C. from the Consortium for Functional Glycomics from the US National Institute of General Medical Sciences (GM62116) and an Exceptional, Unconventional Research Enabling Knowledge Acceleration (EUREKA) grant (GM085448) to D.F.S. from the National Institute of General Medical Sciences. We thank T. Burgess for helpful discussions.

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Authors and Affiliations

  1. Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA

    Xuezheng Song, Yi Lasanajak, Baoyun Xia, Jamie Heimburg-Molinaro, Hong Ju, Chunmei Zhao, Richard D Cummings & David F Smith

  2. Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, USA

    Jeanne M Rhea & Ross J Molinaro

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  1. Xuezheng Song
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Contributions

X.S., R.D.C. and D.F.S. planned the project, and X.S., Y.L., B.X., H.J., C.Z., J.M.R. and R.J.M. carried out the experiments and supplied critical reagents. X.S., Y.L., J.H.-M., R.D.C. and D.F.S. analyzed the data and wrote the manuscript. All authors edited and commented on the manuscript.

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Correspondence to Richard D Cummings or David F Smith.

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The authors declare no competing financial interests.

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Supplementary Figures 1–6 and Supplementary Tables 1–3 (PDF 4778 kb)

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Song, X., Lasanajak, Y., Xia, B. et al. Shotgun glycomics: a microarray strategy for functional glycomics. Nat Methods 8, 85–90 (2011). https://doi.org/10.1038/nmeth.1540

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