Glycobiology Advance Access published online on March 31, 2008
Glycobiology, doi:10.1093/glycob/cwn028
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A Simplified and Sensitive Fluorescent Method for Disaccharide Analysis of both Heparan Sulphate and Chondroitin / Dermatan Sulphates 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, United Kingdom
1 To whom correspondence should be addressed: Tel: 0161 446 3202; Fax: 0161 446 3269; E-mail: MLyon{at}picr.man.ac.uk
Received on February 7, 2008; accepted on March 26, 2008
Sulphated 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 sulphation. Disaccharide analysis of such glycosaminoglycans yields important diagnostic and comparative structural information on sulphate 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 analysing different glycosaminoglycans. We describe a simple procedure, requiring only standard HPLC instrumentation, involving pre-derivatisation of disaccharides with 2-amino-acridone with no clean-up of samples, followed by separation by reverse-phase HPLC, that is sensitive to as little as 
100pg (10–13 mol) of individual disaccharide, thereby allowing analyses of >10ng of total glycosaminoglycan. Importantly, separate analysis of both HS/heparin and CS/DS species within a mixed GAG 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 from MDCK cells (which revealed a HS species of relatively low N-sulphation, but high O- sulphation). This simplified method should find widespread utility for analysing glycosaminoglycans from limited animal and cell culture samples.
Key words: disaccharides / chondroitin sulphate / dermatan sulphate / glycosaminoglycans / heparan sulphate