GLYCOSYL COMPOSTION ANALYSIS OT TWO POMGNT1 SAMPLES (FROM MICE)

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来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 糖基组成分析进行了组合的气相色谱/质谱（GC/MS）的全-O-三甲基甲硅烷基（TMS）衍生物的单糖甲基糖苷的样品通过酸性甲醇分解。 首先从客户提供的200 g干样品中制备甲基糖苷，方法是在80 ℃下在1 M HCl的甲醇溶液中进行甲醇分解（18-22小时），然后在甲醇中用吡啶和乙酸酐进行重新N-乙酰化（用于检测氨基糖）。 然后通过用Tri-Sil（Pierce）在80 ° C下处理（0.5小时），将样品全-O-三甲基甲硅烷基化。这些程序如前所述进行（Merkle，R. K. Poppe，我。等人（1994）酶学方法。230：1-15;约克W.美国，Darvill，A. G.，McNeil，M.，史蒂文森，T. T.，和Albersheim，P.（1985）Methods Enzymol. 118：3-40）。 TMS甲基糖苷的GC/MS分析在与5970 MSD连接的HP 5890 GC上进行，使用Supelco DB-1熔融二氧化硅毛细管柱（30 m x 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. Methyl glycosides were first prepared from 200 ¿g of the dry sample provided by the client by methanolysis in 1 M HCl in methanol at 80¿C (18-22 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 80oC (0.5 hours). These procedures were carried out as previously described (Merkle, R. K. and Poppe, I. (1994) Methods Enzymol. 230:1-15; York W. S., Darvill, A. G., McNeil, M., Stevenson, T. T., and Albersheim, P. (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).