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. The sample was transferred to a screw-cap tube and added with 4 ?g inositol as internal standard, and dried. Methyl glycosides then were prepared from the dried samples by methanolysis with 1 M HCl in methanol at 80?C for 18 h. The preceding step was followed by re-N-acetylation with pyridine and acetic anhydride in methanol (for detection of amino sugars). The samples then were per-O-trimethylsilylated with Tri-Sil (Pierce) at 80?C for 0.5 h. These procedures were carried out as described previously in Merkle and Poppe (1994) Methods Enzymol. 230: 1-15;York, et al. (1985) Methods Enzymol. 118:3-40. GC/MS analysis of the TMS methyl glycosides was performed on a Hewlett Packard Series II 5890 interfaced to a Hewlett Packard 5970 MSD, using a Supelco EC-1 fused silica capillary column (30m ? 0.25 mm ID).
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