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. Glycosyl composition analysis was performed by combined gas chromatography/mass spectrometry (GC/MS) of the per-O-trimethylsilyl (TMS) derivatives of the monosaccharide methyl glycosides produced from the sample by acidic methanolysis. Methyl glycosides were first prepared from 1.0 mg of the dry sample provided by the client by methanolysis in 1 M HCl in methanol at 80 C (18-22 hours), followed by re-N-acetylation with pyridine and acetic anhydride in methanol (for detection of amino sugars). The samples were then per-O-trimethylsilylated by treatment with Tri-Sil (Pierce) at 80 C (0.5 hours). These procedures were carried out as previously described in Merkle and Poppe (1994) Methods Enzymol. 230:1-15 and in York et al. (1985) Methods Enzymol. 118:3-40. GC/MS analysis of the TMS methyl glycosides was performed on an HP 5890 GC interfaced to a 5970 MSD, using a Supelco DB-1 fused silica capillary column (30m x 0.25 mm ID). HPLC was carried out with a ThermoFinnigan Surveyor pump and autosampler and a Restek Pinnacle II C18 chromatography column (5 mm, 250 x 1 mm). Eluent solutions of water/acetonitrile in 0.01% TFA were used to create various gradients, optimizing the separation of Saponins. Flow rates ranged between 50 and 100 mL/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 50 mM solution of oligosaccharides. 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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