This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Heparinase digestion A 75 uL aliquot of a 1.5 g/L solution of low molecular weight heparin (AVT and HSP) in 20 mM ammonium acetate containing 2 mM calcium acetate was treated with 10 uL of a mixture of heparinases I, II, and III (1 U/mL each) and incubated at 37 C. After 20 h, the reaction was quenched by boiling the mixture for 2 min. The samples were filtered (0.2 microns) and 2 uL portions injected into the LC/MS. LC/MS HPLC was carried out with a ThermoFinnigan Surveyor pump and autosampler and a Restek Pinnacle II C18 chromatography column (5 um, 250x1 mm). Two eluent solutions of dibutylammonium acetate in water/methanol or water/acetonitrile were used to create various gradients, optimizing the separation of disaccharides. Flow rates ranged between 50 and 100 uL/min. Mass spectra and LC/MS chromatograms were obtained using a ThermoFinnigan LCQ Advantage quadrupole ion trap instrument in the ESI mode. The instrument was tuned using a 100 uM solution of heparin disaccharide III-S in eluent solution. Thus, the spray voltage used was 4.30 kV, the capillary temperature 200 C, and the sheath gas flow rate at 45 (arbitrary units).
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