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Shotgun glycomics: a microarray strategy for functional glycomics


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.


  1. Varki, A. et al. Essentials of Glycobiology 2nd edn. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2009).

  2. Lowe, J.B. & Marth, J.D. A genetic approach to mammalian glycan function. Annu. Rev. Biochem. 72, 643–691 (2003).

    CAS  Article  Google Scholar 

  3. Freeze, H.H. & Aebi, M. Altered glycan structures: the molecular basis of congenital disorders of glycosylation. Curr. Opin. Struct. Biol. 15, 490–498 (2005).

    CAS  Article  Google Scholar 

  4. Cummings, R.D. The repertoire of glycan determinants in the human glycome. Mol. Biosyst. 5, 1087–1104 (2009).

    CAS  Article  Google Scholar 

  5. Stowell, S.R. et al. Innate immune lectins kill bacteria expressing blood group antigen. Nat. Med. 16, 295–301 (2010).

    CAS  Article  Google Scholar 

  6. Iwamori, M. A new turning point in glycosphingolipid research. Hum. Cell 18, 117–133 (2005).

    Article  Google Scholar 

  7. Avci, F.Y. & Kasper, D.L. How bacterial carbohydrates influence the adaptive immune system. Annu. Rev. Immunol. 28, 107–130 (2010).

    CAS  Article  Google Scholar 

  8. Comelli, E.M. et al. A focused microarray approach to functional glycomics: transcriptional regulation of the glycome. Glycobiology 16, 117–131 (2006).

    CAS  Article  Google Scholar 

  9. Paulson, J.C., Blixt, O. & Collins, B.E. Sweet spots in functional glycomics. Nat. Chem. Biol. 2, 238–248 (2006).

    CAS  Article  Google Scholar 

  10. Song, X. et al. Novel fluorescent glycan microarray strategy reveals ligands for galectins. Chem. Biol. 16, 36–47 (2009).

    CAS  Article  Google Scholar 

  11. Macher, B.A. & Sweeley, C.C. Glycosphingolipids: structure, biological source, and properties. Methods Enzymol. 50, 236–251 (1978).

    CAS  Article  Google Scholar 

  12. Li, Y.T. et al. Preparation of homogenous oligosaccharide chains from glycosphingolipids. Glycoconj. J. 26, 929–933 (2009).

    CAS  Article  Google Scholar 

  13. Luyai, A., Lasanajak, Y., Smith, D.F., Cummings, R.D. & Song, X. Facile preparation of fluorescent neoglycoproteins using p-nitrophenyl anthranilate as a heterobifunctional linker. Bioconjug Chem. 20, 1618–1624 (2009).

    CAS  Article  Google Scholar 

  14. Willison, H.J. Gangliosides as targets for autoimmune injury to the nervous system. J. Neurochem. 103 (Suppl. 1), 143–149 (2007).

    CAS  Article  Google Scholar 

  15. Garcia-Monco, J.C., Seidman, R.J. & Benach, J.L. Experimental immunization with Borrelia burgdorferi induces development of antibodies to gangliosides. Infect. Immun. 63, 4130–4137 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Terabayashi, T. & Kawanishi, Y. Naturally occurring ganglioside lactones in Minke whale brain. Carbohydr. Res. 307, 281–290 (1998).

    CAS  Article  Google Scholar 

  17. Riboni, L. et al. Natural occurrence of ganglioside lactones. Isolation and characterization of GD1b inner ester from adult human brain. J. Biol. Chem. 261, 8514–8519 (1986).

    CAS  PubMed  Google Scholar 

  18. Bassi, R., Riboni, L., Sonnino, S. & Tettamanti, G. Lactonization of GD1b ganglioside under acidic conditions. Carbohydr. Res. 193, 141–146 (1989).

    CAS  Article  Google Scholar 

  19. Anstee, D.J. The nature and abundance of human red cell surface glycoproteins. J. Immunogenet. 17, 219–225 (1990).

    CAS  Article  Google Scholar 

  20. Zhang, G. et al. Suppression of human prostate tumor growth by a unique prostate-specific monoclonal antibody F77 targeting a glycolipid marker. Proc. Natl. Acad. Sci. USA 107, 732–737 (2010).

    CAS  Article  Google Scholar 

  21. Song, X., Lasanajak, Y., Xia, B., Smith, D.F. & Cummings, R.D. Fluorescent glycosylamides produced by microscale derivatization of free glycans for natural glycan microarrays. ACS Chem. Biol. 4, 741–750 (2009).

    CAS  Article  Google Scholar 

  22. Blixt, O. et al. Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc. Natl. Acad. Sci. USA 101, 17033–17038 (2004).

    CAS  Article  Google Scholar 

  23. Muthing, J. Analyses of glycosphingolipids by high-performance liquid chromatography. Methods Enzymol. 312, 45–64 (2000).

    CAS  Article  Google Scholar 

  24. Ohara, K., Sano, M., Kondo, A. & Kato, I. Two-dimensional mapping by high-performance liquid chromatography of pyridylamino oligosaccharides from various glycosphingolipids. J. Chromatogr. A 586, 35–41 (1991).

    CAS  Article  Google Scholar 

  25. Wing, D.R. et al. High-performance liquid chromatography analysis of ganglioside carbohydrates at the picomole level after ceramide glycanase digestion and fluorescent labeling with 2-aminobenzamide. Anal. Biochem. 298, 207–217 (2001).

    CAS  Article  Google Scholar 

  26. Lopez, P.H. & Schnaar, R.L. Determination of glycolipid-protein interaction specificity. Methods Enzymol. 417, 205–220 (2006).

    CAS  Article  Google Scholar 

  27. Magnani, J.L. et al. A monosialoganglioside is a monoclonal antibody-defined antigen of colon carcinoma. Science 212, 55–56 (1981).

    CAS  Article  Google Scholar 

  28. Stoll, M.S. et al. Fluorescent neoglycolipids. Improved probes for oligosaccharide ligand discovery. Eur. J. Biochem. 267, 1795–1804 (2000).

    CAS  Article  Google Scholar 

  29. Laine, R.A., Yoggeswaran, G. & Hakomori, S. Glycosphingolipids covalently linked to agarose gel or glass beads. Use of the compounds for purification of antibodies directed against globoside and hematoside. J. Biol. Chem. 249, 4460–4466 (1974).

    CAS  PubMed  Google Scholar 

  30. Hirabayashi, Y., Hamaoka, A., Matsumoto, M. & Nishimura, K. An improved method for the separation of molecular species of cerebrosides. Lipids 21, 710–714 (1986).

    CAS  Article  Google Scholar 

  31. Song, X., Xia, B., Lasanajak, Y., Smith, D.F. & Cummings, R.D. Quantifiable fluorescent glycan microarrays. Glycoconj. J. 25, 15–25 (2008).

    CAS  Article  Google Scholar 

  32. Xia, B. et al. Versatile fluorescent derivatization of glycans for glycomic analysis. Nat. Methods 2, 845–850 (2005).

    CAS  Article  Google Scholar 

  33. Jones, K.L. et al. Strong IgG antibody responses to Borrelia burgdorferi glycolipids in patients with Lyme arthritis, a late manifestation of the infection. Clin. Immunol. 132, 93–102 (2009).

    CAS  Article  Google Scholar 

  34. Kinjo, Y. et al. Natural killer T cells recognize diacylglycerol antigens from pathogenic bacteria. Nat. Immunol. 7, 978–986 (2006).

    CAS  Article  Google Scholar 

  35. Schnaar, R.L. Isolation of glycosphingolipids. Methods Enzymol. 230, 348–370 (1994).

    CAS  Article  Google Scholar 

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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



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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Song, X., Lasanajak, Y., Xia, B. et al. Shotgun glycomics: a microarray strategy for functional glycomics. Nat Methods 8, 85–90 (2011).

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