COMPOSITION ANALYSIS BY GC-MS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 采用气相色谱/质谱仪(GC/MS)联用技术，对酸解法制备的甲基糖苷的全O-三甲基硅基(TMS)衍生物进行了糖基组成分析。 样品转移到螺帽试管中，加入4g肌醇作为内标，干燥。干燥后的样品在80℃下用1MHCl在甲醇中甲烷化18h，然后在甲醇中与吡啶和乙酸酐进行再N-乙酰化反应(用于氨基糖的检测)。然后，样品在80℃下用Tri-Sill(Piels)进行全O-三甲基硅烷化0.5h。这些步骤按照前面Merkle和Poppe(1994)方法Enzymo.230：1-15；York等人。(1985)方法Enzyol。118：3-40。TMS甲基糖苷的GC/MS分析在Hewlett Packard Series II 5890与Hewlett Packard 5970 MSD接口，使用Supelco EC-1熔融石英毛细管柱(30m×0.25 mm ID)。

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).