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High-performance liquid chromatography-mass spectrometry for mapping and sequencing glycosaminoglycan-derived oligosaccharides

Nature Protocols volume 5, pages 993–1004 (2010)Cite this article

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Abstract

Glycosaminoglycans (GAGs) have proven to be very difficult to analyze and characterize because of their high negative charge density, polydispersity and sequence heterogeneity. As the specificity of the interactions between GAGs and proteins results from the structure of these polysaccharides, an understanding of GAG structure is essential for developing a structure–activity relationship. Electrospray ionization (ESI) mass spectrometry (MS) is particularly promising for the analysis of oligosaccharides chemically or enzymatically generated by GAGs because of its relatively soft ionization capacity. Furthermore, on-line high-performance liquid chromatography (HPLC)-MS greatly enhances the characterization of complex mixtures of GAG-derived oligosaccharides, providing important structural information and affording their disaccharide composition. A detailed protocol for producing oligosaccharides from various GAGs, using controlled, specific enzymatic or chemical depolymerization, is presented, together with their HPLC separation, using volatile reversed-phase ion-pairing reagents and on-line ESI-MS structural identification. This analysis provides an oligosaccharide map together with sequence information from a reading frame beginning at the nonreducing end of the GAG chains. The preparation of oligosaccharides can be carried out in 10 h, with subsequent HPLC analysis in 1–2 h and HPLC-MS analysis taking another 2 h.

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Figure 1: Different classes of GAGs and their disaccharide units.
Figure 2: SAX-HPLC of HA oligosaccharides produced by testicular hyaluronidase digestion detected at 214 nm.
Figure 3: RPIP-HPLC separation of HP unsaturated oligosaccharides obtained from controlled heparinase I depolymerization.
Figure 4: Total ion chromatograms of Scapharca inaequivalvis DS oligosaccharides up to 10-mer in the negative ion mode obtained by partial treatment with chondroitin ABC lyase and separated by means of RPIP-HPLC.
Figure 5: The ESI-MS spectrum in the negative mode of each single Scapharca inaequivalvis DS oligosaccharide species separated by means of RPIP-HPLC from 1- to 10-mer.
Figure 6: Total ion chromatograms of K4 oligosaccharides up to 24-mers in negative ion mode separated by means of RPIP-HPLC.
Figure 7: Total ion chromatograms of unsulfated saturated chondroitin oligosaccharides up to 16-mers in negative ion mode separated by means of RPIP-HPLC.
Figure 8: Total ion chromatograms of HA-saturated oligosaccharides up to 40-mers in negative ion mode separated by means of RPIP-HPLC.
Figure 9: Analysis of unsaturated oligosaccharides derived from nonsulfated HA and HN GAGs.
Figure 10: MS/MS spectra of HA 4-mer, tetrasaccharide from HA and HN 4-mer, tetrasaccharide from HN.

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Acknowledgements

This work was supported in part through grants from the US National Institutes of Health (GM38060, HL096972, HL101721 and GM090127) to R.J.L.

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Authors and Affiliations

  1. Department of Biology, University of Modena and Reggio Emilia, Modena, Italy

    Nicola Volpi

  2. Departments of Chemistry and Chemical Biology, Chemical and Biological Engineering and Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, USA

    Robert J Linhardt

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All authors contributed equally to this work. R.J.L. designed and developed the RPIP-HPLC-ESI-MS. R.J.L. and N.V. applied this methodology to the structural study of various complex natural polymers with respect to bacterial polysaccharides.

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Correspondence to Nicola Volpi.

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Volpi, N., Linhardt, R. High-performance liquid chromatography-mass spectrometry for mapping and sequencing glycosaminoglycan-derived oligosaccharides. Nat Protoc 5, 993–1004 (2010). https://doi.org/10.1038/nprot.2010.48

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