Disaccharide compositional analysis of heparan sulfate and heparin polysaccharides using UV or high-sensitivity fluorescence (BODIPY) detection

One of the first steps in characterizing heparan sulfate (HS) and its close relative heparin is to conduct disaccharide composition analysis. This provides an overall picture of the structure of the polysaccharide in terms of its constituent disaccharides. This is of importance, for example, in the...

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Veröffentlicht in:Nature protocols Jg. 5; H. 12; S. 1983 - 1992
Hauptverfasser: Skidmore, Mark A, Guimond, Scott E, Dumax-Vorzet, Audrey F, Yates, Edwin A, Turnbull, Jeremy E
Format: Journal Article
Sprache:Englisch
Veröffentlicht: London Nature Publishing Group UK 01.12.2010
Nature Publishing Group
Schlagworte:
631/1647/2230/1378
631/1647/666/2260
631/45/72/1205
Analytical Chemistry
Anticoagulants
Biological Techniques
Biomedical and Life Sciences
Boron Compounds
Carbohydrates
Chromatography, High Pressure Liquid - methods
Composition
Computational Biology/Bioinformatics
Disaccharides - isolation & purification
Enzymes
Fluorescence
Fluorescent Dyes
Heparan sulfate
Heparin
Heparin - analysis
Heparitin Sulfate - analysis
High performance liquid chromatography
Life Sciences
Liquid chromatography
Methods
Microarrays
Organic Chemistry
Properties
protocol
Saccharides
Spectrophotometry, Ultraviolet - methods
Sugars
Sulfates
United Kingdom
United Kingdom > UK
ISSN:1754-2189, 1750-2799, 1750-2799
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Zusammenfassung:One of the first steps in characterizing heparan sulfate (HS) and its close relative heparin is to conduct disaccharide composition analysis. This provides an overall picture of the structure of the polysaccharide in terms of its constituent disaccharides. This is of importance, for example, in the initial characterization of spatially and temporally regulated structures. Two protocols for conducting disaccharide analysis are presented here, both exploiting exhaustive digestion of the polysaccharide, yielding constituent disaccharides, by bacterial heparin lyases. The first method, suitable for microgram quantities of material, relies on the separation of the disaccharides by high-performance liquid chromatography (HPLC) coupled to ultraviolet absorbance detection and can be performed in 2 d. The second exploits reducing end–labeling with the fluorophore BODIPY hydrazide, separation by HPLC, and subsequent fluorescence detection and quantitation. The latter is a high-sensitivity method that requires nanograms of starting material and has a detection limit in the low fmol range, and is thus the most sensitive method for disaccharide compositional analysis of HS yet reported. Fluorescence detection can be routinely carried out in 3 d.
Bibliographie:ObjectType-Article-1
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ISSN:1754-2189
1750-2799
1750-2799
DOI:10.1038/nprot.2010.145