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. 2017 Feb;34(1):107-117.
doi: 10.1007/s10719-016-9734-7. Epub 2016 Oct 22.

An integrated approach using orthogonal analytical techniques to characterize heparan sulfate structure

Affiliations

An integrated approach using orthogonal analytical techniques to characterize heparan sulfate structure

Daniela Beccati et al. Glycoconj J. 2017 Feb.

Abstract

Heparan sulfate (HS), a glycosaminoglycan present on the surface of cells, has been postulated to have important roles in driving both normal and pathological physiologies. The chemical structure and sulfation pattern (domain structure) of HS is believed to determine its biological function, to vary across tissue types, and to be modified in the context of disease. Characterization of HS requires isolation and purification of cell surface HS as a complex mixture. This process may introduce additional chemical modification of the native residues. In this study, we describe an approach towards thorough characterization of bovine kidney heparan sulfate (BKHS) that utilizes a variety of orthogonal analytical techniques (e.g. NMR, IP-RPHPLC, LC-MS). These techniques are applied to characterize this mixture at various levels including composition, fragment level, and overall chain properties. The combination of these techniques in many instances provides orthogonal views into the fine structure of HS, and in other instances provides overlapping / confirmatory information from different perspectives. Specifically, this approach enables quantitative determination of natural and modified saccharide residues in the HS chains, and identifies unusual structures. Analysis of partially digested HS chains allows for a better understanding of the domain structures within this mixture, and yields specific insights into the non-reducing end and reducing end structures of the chains. This approach outlines a useful framework that can be applied to elucidate HS structure and thereby provides means to advance understanding of its biological role and potential involvement in disease progression. In addition, the techniques described here can be applied to characterization of heparin from different sources.

Keywords: Analytical; Domains; HPLC; Heparan sulfate; MS; NMR; Structure.

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Conflict of interest statement

All authors are or at some point were employees of Momenta Pharmaceuticals Inc. with stock compensation. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

Figures

Fig. 1
Fig. 1
a 1H–NMR and b HSQC spectra of BKHS. Signals due to the reducing end (xyl α/β and HNAc α), non-reducing end (G n.r.), and differently substituted internal I2S are indicated

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