Generating heparan sulfate saccharide libraries for glycomics applications

Centre for Glycobiology, School of Biological Sciences, University of Liverpool, Liverpool, UK.
Nature Protocol (Impact Factor: 9.67). 05/2010; 5(5):821-33. DOI: 10.1038/nprot.2010.17
Source: PubMed


Natural and semi-synthetic heparan sulfate (HS) saccharide libraries are a valuable resource for investigating HS structure-function relationships, enabling high-throughput glycomics studies. Owing to the difficulty of chemical or in vitro enzymatic synthesis of HS saccharides, the structural diversity displayed in saccharides from tissue or cell sources cannot be readily accessed. In contrast, saccharide libraries can be generated by partial digestion of tissue-derived HS polysaccharide chains and chromatographic fractionation of the resulting saccharide mixtures. Fractionation is initially on the basis of hydrodynamic volume, using size exclusion chromatography. Further fractionation, on the basis of charge using strong anion exchange, can subsequently be applied. Desalting and sample concentration follows each fractionation step. Chromatographic fractions are generated that contain purified, or partially purified, saccharides. Here we describe a comprehensive protocol for generation of structurally diverse natural saccharide libraries from HS variants that is fast (approximately 3 weeks) and reproducible.

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Available from: Edwin Alexander Yates
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    • "The same heparin (17 kDa average molecular weight) (Celsus Lab, Cincinnati, OH, USA) was used throughout, including for the production of the chemically modified polysaccharides (Table 1) and oligosaccharides of defined size. Heparin derivatives were as characterised previously (Yates et al., 1996) and heparin derived oligosaccharides were obtained from partial heparinase I (IBEX Technologies, Montreal, Canada) digestion of porcine heparin performed, as described previously (Popplewell et al., 2009; Powell et al., 2010). "
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    • "Purified oligosaccharides were analyzed by PAGE and quantified by weighing or by measuring their absorption at 232 nm. Compositional analysis was used to confirm the presence of the major disaccharide repeat of the trisulfated disaccharide UA(2S)-GlcNS(6S) (Powell et al, 2010). "
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