Summary
This chapter describes two methods for fabricating microarrays of saccharides for display and interrogation with binding proteins, using fluorescence detection. The first approach is based on the rapid immobilization of heparan sulphate glycans upon commercially available aminosilane slides via their reducing ends. The second approach is based on the use of a hydrazide-derivatized self-assembled monolayer (SAM) on a gold-coated slide surface. Both provide for efficient and chemoselective attachment and anchoring of oligosaccharide probes via their reducing ends, enabling the large-scale arraying of natural saccharides without cumbersome pre-derivatization. The latter platform, in particular, also has the potential for use with other biophysical readout methods including matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, surface plasmon resonance, and quartz crystal microbalances. These microarray platforms provide a facile approach for interrogating multiple carbohydrate-protein interactions in a high-throughput manner using minimal quantities of reagents. They provide an essential new experimental strategy in the growing armoury of the glycomics toolkit.
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References
Shin I, Park S, Lee MR. (2005). Carbohydrate microarrays: an advanced technology for functional studies of glycans. Chemistry 11, 2894–2901
Ratner DM, Adams EW, Disney MD, Seeberger PH (2004). Tools for glycomics: mapping interactions of carbohydrates in biological systems. ChemBiochem 5, 1375–1383
Zhi ZL, Powell AK, Turnbull JE (2006). Fabrication of carbohydrate microarray on gold surface: direct attachment of non-derivatized oligosaccharides to hydrazide-derivatized self-assembled monolayer. Anal. Chem. 78, 4786–4793
Powell AK, Fernig DG, Turnbull JE (2002). Fibroblast growth factor receptors 1 and 2 interact differently with heparin/heparan sulfate – implications for dynamic assembly of a ternary signaling complex. J. Biol. Chem. 277, 28554–28563
Yates EA, Jones MO, Clarke C, Powell AK, Johnson SR, Porch A, Edwards P, Turnbull JE (2004). Microwave enhanced reaction of carbohydrates with amino-derivatised labels and glass surfaces. J. Mater. Chem. 13, 2061–2063
Skidmore MA, Turnbull JE (2005). Separation & sequencing of heparin and HS saccharides. In: Chemistry and Biology of Heparin and Heparan Sulphate, Garg H (ed.). Elsevier Press, New York, pp. 181–203
Linhardt RJ, Turnbull JE, Wang H, Loganathan D, Gallagher JT (1990). Examination of the substrate specificity of heparin and heparan sulphate lyases. Biochemistry 29, 2611–2617
Duchesne L, Tissot B, Rudd TR, Dell A, Fernig DG (2006). N-glycosylation of fibroblast growth factor receptor 1 regulates ligand and heparan sulfate co-receptor binding. J Biol Chem 281, 27178–27189
Flinn NS, Quibell M, Monk TP, Ramjee MK, Urch CJ (2006). A single-step method for the production of sugar hydrazides: Intermediates for the chemoselective preparation of glycoconjugates. Bioconjug. Chem. 16, 722–728, 2005
Acknowledgements
This research was supported by the UK ‘Glycoarrays’ Consortium funded by the Research Councils UK Basic Technology Programme (to JET); a Biotechnology and Biological Sciences Research Council project grant (to AKP and JET) and a grant to the Liverpool Centre for Bioarray Innovation (Prof. Andy Cossins), and a Medical Research Council Senior Research Fellowship (to JET). The Authors would like to thank Yassir Ahmed, and Lorraine Wallace for technical assistance in the production of saccharides and PAGE analysis, Abdelmadjid Atrih for mass spectrometry analysis of saccharides and Margaret Hughes for instruction and advice in operation of the microarray robot. We also thank Rob Field, Jonathan Blackburn, Marianne Brille, Tim Rudd, Ed Yates and Dave Fernig for useful discussions.
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Powell, A., Zhi, Zl., Turnbull, J. (2009). Saccharide Microarrays for High-Throughput Interrogation of Glycan-Protein Binding Interactions. In: Packer, N.H., Karlsson, N.G. (eds) Glycomics. Methods in Molecular Biology™, vol 534. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-022-5_22
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DOI: https://doi.org/10.1007/978-1-59745-022-5_22
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