GLYCOSYL COMPOSITION ANALYSIS OF TWO SAMPLES BY GC-MS

这个子项目是利用资源的许多研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 糖基组成（GC-MS法） 将样品转移至预冲洗的螺旋盖管中，加入3 μ g肌醇作为内标，并冻干。 然后，通过用1 M HCl的甲醇溶液在80 ℃下甲醇分解过夜，然后用吡啶和乙酸酐的甲醇溶液进行再N-乙酰化（用于检测氨基糖），从干燥的样品制备甲基糖苷。 将样品的甲基糖苷用Tri-Sil试剂（Thermo Scientific）在80 ℃下全-O-三甲基甲硅烷基化（TMS）0.5小时。这些程序如先前在Merkle和Poppe（1994）Methods Enzymol. 230：1-15;约克等人（1985）酶学方法（Methods Enzymol. 118：3-40。TMS甲基糖苷的分析在配备有Supelco EC-1熔融石英毛细管柱（30 m × 0.25 mm ID）并与Hewlett Packard 5970 MSD连接的Hewlett Packard Series II 5890气相色谱仪上进行。 由于预计分析物的绝对含量较低，因此将最终衍生化样品溶于少量己烷中，必须手动进样。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Glycosyl composition by GC-MS The sample was transferred to a pre-rinsed screw-cap tube, added with 3 ¿g of inositol as an internal standard, and lyophilized. Methyl glycosides then were prepared from the dried sample by methanolysis with 1 M HCl in methanol at 80¿C overnight followed by re-N-acetylation with pyridine and acetic anhydride in methanol (for detection of amino sugars). The methyl glycosides of the sample were per-O-trimethylsilylated (TMS) with a Tri-Sil reagent (Thermo Scientific) 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. Analysis of the TMS methyl glycosides was performed on a Hewlett Packard Series II 5890 gas chromatograph equipped with a Supelco EC-1 fused silica capillary column (30m ¿ 0.25 mm ID) and interfaced to a Hewlett Packard 5970 MSD. Since the absolute content of the analytes was expected to be low, the final derivatized sample was dissolved in a small volume of hexane and had to be injected manually.