High sensitivity separation and detection of heparan sulfate disaccharides.
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.
Skidmore
M A
MA
School of Biological Sciences, University of Liverpool, Crown Street, Liverpool L69 7ZB, UK.
Guimond
S E
SE
Dumax-Vorzet
A F
AF
Atrih
A
A
Yates
E A
EA
Turnbull
J E
JE
D001896:Boron Compounds / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D004187:Disaccharides / Q000032:analysis* / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006497:Heparan Sulfate / Q000032:analysis* / Q000737:chemistry; D015203:Reproducibility of Results; D012492:Salts / Q000737:chemistry; D012680:Sensitivity and Specificity; D012972:Sodium Hydroxide / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
D001896:Boron Compounds; D004187:Disaccharides; D005456:Fluorescent Dyes; D006497:Heparan Sulfate; D012492:Salts; D012972:Sodium Hydroxide; C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene