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 prepared from 0.8 mg of the dry sample by methanolysis in 1 M HCl in methanol at 80 C (18 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 (20 min). 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). MALDI-TOF-MS was performed with a Voyager mass spectrometer operated in the positive ion mode. The mass spectrometer was calibrated with a mixture of maltooligosaccharides. Aqueous solutions of samples were diluted 1:1 with 2,5-dihydroxybenzoic acid (DHB) and a portion (1.0 ul) was applied to the sample plate of the MS. Samples were desorbed from the sample plate with a nitrogen laser having a guide wire reading of 0.05% and a laser intensity of approximately 2100. NMR spectroscopy was performed by the sample being deuterium-exchanged and lyophilized from D2O and dissolved in 0.5 mL pyridine-d5. 1- and 2-D NMR spectra were acquired on a Varian Inova-600 MHz spectrometer at 298 K (25 C). Proton chemical shifts were measured relative to the most upfield pyridine-d5 singlet delta=7.22 ppm).
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