Labelling heparan sulphate saccharides with chromophore, fluorescence and mass tags for HPLC and MS separations.
The analysis of heparin/HS saccharides derived from small amounts of tissue or cells is a considerable technical challenge and the development of methods to characterise these carbohydrates has progressed comparatively slowly. A number of procedures have been devised to tag glycans selectively at the reducing end with a group that will enhance the sensitivity of detection and facilitate chromatographic separations. Outlined in this chapter are two useful strategies designed specifically for the analysis of heparin/HS saccharides. The first involves a fluorophore label, Bodipy-FL-hydrazide, which permits highly sensitive (fmol level) detection of saccharides utilising high performance strong anion exchange chromatography. The second facilitates oligosaccharide separation by gel-permeation chromatography and reverse phase high performance ion-pairing chromatography (RP-HPIPC) through the use of a phenylsemicarbazide tag. The latter also serves as an effective mass tag for electrospray mass spectrometry, permitting enhanced analysis of HS saccharides. These methods provide new opportunities for the development of glycomics approaches to study the structure and function of the heparan sulfate family of glycans.
Skidmore
Mark
M
School of Biological Sciences, University of Liverpool, Liverpool, UK.
Atrih
Abdel
A
Yates
Ed
E
Turnbull
Jeremy E
JE
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002863:Chromogenic Compounds / Q000494:pharmacology*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006497:Heparan Sulfate / Q000032:analysis / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008954:Models, Biological; D008970:Molecular Weight; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry; D013194:Staining and Labeling / Q000379:methods*
D002863:Chromogenic Compounds; D005456:Fluorescent Dyes; D006497:Heparan Sulfate; D009844:Oligosaccharides