COMPOSITION ANALYSIS BY GC-MS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 将样品转移至螺旋盖管中，加入2 μ g肌醇作为内标，并冻干。 在80 ° C下，用甲醇中的1 M HCl对样品进行甲醇分解过夜以形成甲基糖苷。 水解后，将样品在氮气流下完全干燥。 在室温下，在甲醇中用吡啶和乙酸酐将甲基糖苷重新N-乙酰化（用于检测氨基糖）至少30 min。然后在80 ℃下用Tri-Sil试剂（Thermo Scientific）将样品全-O-三甲基甲硅烷基化（TMS）20 min。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。

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. The samples were transferred into screw-cap tubes, added with 2 ¿g inositol as internal standard, and lyophilized. The samples were subjected to methanolysis with 1 M HCl in methanol at 80¿C overnight to form methyl glycosides. After hydrolysis, the samples were dried completely under a stream of nitrogen gas. The methyl glycosides were re-N-acetylated with pyridine and acetic anhydride in methanol (for detection of amino sugars) at room temperature for at least 30 min. The samples then were per-O-trimethylsilylated (TMS) with a Tri-Sil reagent (Thermo Scientific) at 80¿C for 20 min. 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.