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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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.
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). https://doi.org/10.1038/nmeth.1540
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