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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Vydáno v:	Nature protocols Ročník 5; číslo 12; s. 1983 - 1992
Hlavní autoři:	Skidmore, Mark A, Guimond, Scott E, Dumax-Vorzet, Audrey F, Yates, Edwin A, Turnbull, Jeremy E
Médium:	Journal Article
Jazyk:	angličtina
Vydáno:	London Nature Publishing Group UK 01.12.2010 Nature Publishing Group
Témata:	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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Abstract	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.
AbstractList	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. 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.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.
Audience	Academic
Author	Yates, Edwin A Dumax-Vorzet, Audrey F Turnbull, Jeremy E Guimond, Scott E Skidmore, Mark A
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Snippet	One of the first steps in characterizing heparan sulfate (HS) and its close relative heparin is to conduct disaccharide composition analysis. This provides an...
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SubjectTerms	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
Title	Disaccharide compositional analysis of heparan sulfate and heparin polysaccharides using UV or high-sensitivity fluorescence (BODIPY) detection
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