High sensitivity separation and detection of heparan sulfate disaccharides |
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Authors: | Skidmore M A Guimond S E Dumax-Vorzet A F Atrih A Yates E A Turnbull J E |
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Institution: | School of Biological Sciences, University of Liverpool, Crown Street, Liverpool L69 7ZB, UK. |
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Abstract: | Eight Delta-disaccharide standards from heparan sulfate/heparin were derivatized with the fluorophore 4,4-difluoro-5,7- dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, hydrazide (BODIPY) via formation of a Schiff's base and separated using HPAEC on a Propac PA1 column with a linear salt gradient and isocratic 150 mM NaOH. Detection was with an in-line fluorescence detector. The standard deviation (sigma(n-1)) in retention times were 0.7-2% over nine runs. The limit of detection, was 100 fmol (100 x 10(-15)mol) of BODIPY labeled Delta-disaccharides, representing considerably improved detection compared to other fluorophore labeled derivatives and, unlike these, required no further purification steps. Separation and improved detection of BODIPY-Delta-disaccharide conjugates will assist the structural analysis of HS and the development of improved sequencing methodologies. |
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