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Disaccharide compositional analysis of heparan sulfate and heparin polysaccharides using UV or high-sensitivity fluorescence (BODIPY) detection

Nature Protocols volume 5, pages 1983–1992 (2010)Cite this article

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Abstract

One of the first steps in characterizing heparan sulfate (HS) and its close relative heparin is to conduct disaccharide composition analysis. This provides an overall picture of the structure of the polysaccharide in terms of its constituent disaccharides. This is of importance, for example, in the initial characterization of spatially and temporally regulated structures. Two protocols for conducting disaccharide analysis are presented here, both exploiting exhaustive digestion of the polysaccharide, yielding constituent disaccharides, by bacterial heparin lyases. The first method, suitable for microgram quantities of material, relies on the separation of the disaccharides by high-performance liquid chromatography (HPLC) coupled to ultraviolet absorbance detection and can be performed in 2 d. The second exploits reducing end–labeling with the fluorophore BODIPY hydrazide, separation by HPLC, and subsequent fluorescence detection and quantitation. The latter is a high-sensitivity method that requires nanograms of starting material and has a detection limit in the low fmol range, and is thus the most sensitive method for disaccharide compositional analysis of HS yet reported. Fluorescence detection can be routinely carried out in 3 d.

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Figure 1
Figure 2: Structure of the commonly occurring Δ-disaccharides derived from digestion with heparinases I, II and III.
Figure 3: UV SAX HPLC of the eight commonly occurring Δ-disaccharide constituents present within heparin and HS.
Figure 4: Fluorescence SAX HPLC of the eight commonly occurring Δ-disaccharide constituents present within heparin and HS after BODIPY derivatization.
Figure 5: Fluorescence SAX HPLC of the Δ-disaccharide constituents present within porcine mucosa intestinal heparin, exhaustively digested using heparinases I, II and III before derivatization with BODIPY hydrazide.
Figure 6: UV SAX HPLC of the Δ-disaccharide constituents present within the pharmaceutical-grade, low-molecular-weight heparins (LMWH) enoxaparin and tinzaparin.

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Acknowledgements

This study was funded by grants from the Biotechnology and Biological Sciences Research Council (J.E.T., M.A.S., S.E.G. and E.A.Y.), the Wellcome Trust (J.E.T., M.A.S., S.E.G. and E.A.Y.), the Human Frontier Science Program (J.E.T.), the Royal Society (E.A.Y. and M.A.S.) and the Medical Research Council (Senior Research Fellowship to J.E.T.).

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Authors and Affiliations

  1. Centre for Glycobiology, Institute of Integrative Biology, University of Liverpool, Liverpool, UK

    Mark A Skidmore, Scott E Guimond, Edwin A Yates & Jeremy E Turnbull

  2. School of Medicine, University of Manchester, Manchester, UK

    Audrey F Dumax-Vorzet

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Contributions

M.A.S. developed the fluorescence labeling methodology and associated SAX HPLC separation, performed experiments and wrote the article. J.E.T. developed the SAX HPLC methodology for UV detection. S.E.G. and A.F.D. performed labeling and SAX experiments. J.E.T. and E.A.Y. obtained funding, edited the article and provided overall supervision.

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Correspondence to Jeremy E Turnbull.

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Skidmore, M., Guimond, S., Dumax-Vorzet, A. et al. Disaccharide compositional analysis of heparan sulfate and heparin polysaccharides using UV or high-sensitivity fluorescence (BODIPY) detection. Nat Protoc 5, 1983–1992 (2010). https://doi.org/10.1038/nprot.2010.145

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