IMPROVEMENTS IN PROCEDURES FOR PER-O-METHYLATION OF CARBOHYDRATES

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。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 正在开发改进的碳水化合物的Per-O-甲基化方法，这与我们详细研究了在碱存在下二甲基亚砜与甲基碘衍生化过程中可能发生的氧化降解机理的结果一致。我们证明，只有在无水条件下，溶解在二甲基亚砜中的碳水化合物与甲基碘反应时间较长，然后与碱反应，才会发生降解。我们发现，在无水条件下，在引入甲基碘之前用氢氧化钠粉末处理碳水化合物，或者在甲基碘之前在二甲基亚砜中加入微量水，或者使用N，N-二甲基乙酰胺作为溶剂(Ciucanu和Costello，J.Am)，可以完全避免这种情况化学。SoC。，2003年，125125,16213-16219)。后来由其他人(Novotnoy等人，ASMS 2005)报道的利用这种化学洞察力的结果表明，使用填充固体NaOH的毛细管进行微规模反应是可行的。我们已经评估了这种方法，以及其他可能有助于提高效率并将低样本量的污染降至最低的步骤。我们还参与了一组O-连接糖肽样本的循环分析，这些样本是由设在日本大阪的人类糖蛋白组学倡议赞助的一个项目中的几个实验室分析的。描述这一点的一篇手稿最近发表在《分子和细胞蛋白质组学》上。

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. Improved procedures for per-O-methylation of carbohydrates are being developed, consistent with our results from detailed investigation of the possible oxidative degradation mechanisms occurring during the derivatization in dimethyl sulfoxide with methyl iodide in the presence of base. We demonstrated that degradation occurs only under anhydrous conditions when there is a long reaction time between the carbohydrate dissolved in dimethyl sulfoxide and methyl iodide, followed by reaction with the base. We found that it can be totally avoided by treating the carbohydrate with powdered sodium hydroxide before introduction of methyl iodide under non-anhydrous conditions, or by adding a trace of water in dimethyl sulfoxide before methyl iodide, or by using N, N-dimethyl acetamide as the solvent (Ciucanu and Costello, J. Am. Chem. Soc. , 2003, 125, 16213-16219). Later results reported by others (Novotnoy et al., ASMS 2005) that took advantage of this chemical insight have demonstrated that microscale reactions are feasible using capillaries packed with solid NaOH. We have evaluated this approach, and other steps that may help to improve efficiency and minimize contamination for low amounts of sample. We also participated in a round-robin analysis of a set of O-linked glycopeptide samples that were analyzed by several laboratories within a project sponsored by the Human Glycoproteomics Initiative, based in Osaka, Japan. A manuscript describing this was recently published in Molecular and Cellular Proteomics.