A simplified and sensitive fluorescent method for disaccharide analysis of both heparan sulfate and chondroitin/dermatan sulfates from biological samples.

2.50
Hdl Handle:
http://hdl.handle.net/10541/67941
Title:
A simplified and sensitive fluorescent method for disaccharide analysis of both heparan sulfate and chondroitin/dermatan sulfates from biological samples.
Authors:
Deakin, Jon A; Lyon, Malcolm
Abstract:
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.
Affiliation:
Cancer Research UK Glyco-Oncology Group, School of Cancer and Imaging Sciences, University of Manchester, Paterson Institute for Cancer Research, Manchester M20 4BX, UK.
Citation:
A simplified and sensitive fluorescent method for disaccharide analysis of both heparan sulfate and chondroitin/dermatan sulfates from biological samples. 2008, 18 (6):483-91 Glycobiology
Journal:
Glycobiology
Issue Date:
Jun-2008
URI:
http://hdl.handle.net/10541/67941
DOI:
10.1093/glycob/cwn028
PubMed ID:
18378523
Type:
Article
Language:
en
ISSN:
1460-2423
Appears in Collections:
All Paterson Institute for Cancer Research; Medical Oncology

Full metadata record

DC FieldValue Language
dc.contributor.authorDeakin, Jon A-
dc.contributor.authorLyon, Malcolm-
dc.date.accessioned2009-05-12T16:06:41Z-
dc.date.available2009-05-12T16:06:41Z-
dc.date.issued2008-06-
dc.identifier.citationA simplified and sensitive fluorescent method for disaccharide analysis of both heparan sulfate and chondroitin/dermatan sulfates from biological samples. 2008, 18 (6):483-91 Glycobiologyen
dc.identifier.issn1460-2423-
dc.identifier.pmid18378523-
dc.identifier.doi10.1093/glycob/cwn028-
dc.identifier.urihttp://hdl.handle.net/10541/67941-
dc.description.abstractSulfated 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.en
dc.language.isoenen
dc.subject.meshAnimals-
dc.subject.meshCell Line-
dc.subject.meshChondroitin Sulfates-
dc.subject.meshChromatography, High Pressure Liquid-
dc.subject.meshDermatan Sulfate-
dc.subject.meshDisaccharides-
dc.subject.meshDogs-
dc.subject.meshFluorometry-
dc.subject.meshHeparitin Sulfate-
dc.subject.meshLiver-
dc.subject.meshRats-
dc.titleA simplified and sensitive fluorescent method for disaccharide analysis of both heparan sulfate and chondroitin/dermatan sulfates from biological samples.en
dc.typeArticleen
dc.contributor.departmentCancer Research UK Glyco-Oncology Group, School of Cancer and Imaging Sciences, University of Manchester, Paterson Institute for Cancer Research, Manchester M20 4BX, UK.en
dc.identifier.journalGlycobiologyen
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