A simplified and sensitive fluorescent method for disaccharide analysis of both heparan sulfate and chondroitin/dermatan sulfates from biological samples.
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 approximately 100 pg (approximately 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.
Deakin
Jon A
JA
Cancer Research UK Glyco-Oncology Group, School of Cancer and Imaging Sciences, University of Manchester, Paterson Institute for Cancer Research, Manchester M20 4BX, UK.
Lyon
Malcolm
M
D000818:Animals; D002460:Cell Line; D002809:Chondroitin Sulfates / Q000032:analysis* / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003871:Dermatan Sulfate / Q000032:analysis* / Q000737:chemistry; D004187:Disaccharides / Q000032:analysis* / Q000737:chemistry; D004285:Dogs; D005470:Fluorometry / Q000379:methods; D006497:Heparan Sulfate / Q000032:analysis* / Q000737:chemistry; D008099:Liver / Q000737:chemistry; D051381:Rats
D002809:Chondroitin Sulfates; D003871:Dermatan Sulfate; D004187:Disaccharides; D006497:Heparan Sulfate