Glycobiology Advance Access originally published online on March 31, 2008
Glycobiology 2008 18(6):483-491; doi:10.1093/glycob/cwn028
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A simplified and sensitive fluorescent method for disaccharide analysis of both heparan sulfate and chondroitin/dermatan sulfates from biological samples
Cancer Research UK Glyco-Oncology Group, School of Cancer and Imaging Sciences, University of Manchester, Paterson Institute for Cancer Research, Manchester M20 4BX, UK
1 To whom correspondence should be addressed: Tel: +44-0161-446-3202; Fax: +44-0161-446-3269; e-mail: mlyon{at}picr.man.ac.uk
Received on February 7, 2008; revised on March 13, 2008; accepted on March 26, 2008
Sulfated glycosaminoglycans regulate the biological functions of a wide variety of proteins, primarily through high affinity interactions mediated by specific sugar sequences or patterns/densities of sulfation. Disaccharide analysis of such glycosaminoglycans yields important diagnostic and comparative structural information on sulfate patterning. When applied to specific oligosaccharides it can also make a vital contribution to sequence elucidation. Standard UV detection of lyase-generated disaccharides resolved by HPLC can lack sufficient sensitivity and be compromised by contaminating UV signals, when dealing with scarce tissue- or cell culture-derived material. Various methods exist for improved detection, but usually involve additional HPLC hardware and often necessitate different procedures for analyzing different glycosaminoglycans. We describe a simple procedure, requiring only standard HPLC instrumentation, involving prederivatization of disaccharides with 2-aminoacridone with no cleanup of samples, followed by a separation by reverse-phase HPLC that is sensitive to as little as 
100 pg (10–13 mol) of an individual disaccharide, thereby allowing analyses of >10 ng of total glycosaminoglycan. Importantly, separate analysis of both HS/heparin and CS/DS species within a mixed glycosaminoglycan pool can be performed using the same procedure on a single column. We demonstrate its applicability in dealing with small quantities of material derived from rat liver (where we demonstrate a high abundance of the unusual CS-E species within the CS/DS pool) and MDCK cells (which revealed a HS species of relatively low N-sulfation, but high O-sulfation). This simplified method should find a widespread utility for analyzing glycosaminoglycans from limited animal and cell culture samples.
Key words: Chondroitin sulfate / dermatan sulfate / disaccharides / glycosaminoglycans / heparan sulfate