HYBRID TANDEM MASS SPECTROMETRY OF PEPTIDE CONJUGATES

许多外源性物质通过形成反应性亲电 中间体和随后与谷胱甘肽的缀合，容易产生 排泄产物。 这种解毒途径可能会受到损害， 与谷胱甘肽的缀合是无效的，或者如果谷胱甘肽缀合物 自身代谢为亲电产物。 在这些情况下， 毒性作用可由异生物质与细胞蛋白质的结合引起。 谷胱甘肽和蛋白缀合物代谢物的表征可以 提供亲电子中间体的结构信息， 有助于理解结构-毒性关系。 这 是一个特别需要分析的问题，因为 亲电物质可能代表数量较少的代谢途径 因为结构表征必须在 过量的从生物样品中共提取的其他组分。 我们已经开发了一种用于筛选和表征 基于使用稳定同位素标记的谷胱甘肽缀合物， 快原子轰击/串联质谱中的多扫描模式 (MS/MS和MS/MS/MS）。 这种方法允许直接分析复杂的 生物提取物，样品制备最少， 未能观察到目标代谢物的风险较低。 耦合液 色谱-电喷雾电离/串联质谱法提供了一种 一种特别有价值的补充办法， 次要谷胱甘肽结合物代谢物的定量表征。 这些技术将应用于谷胱甘肽结合物的分析 （在体内和体外形成）的几个取代的呋喃，一类 这些化合物显示出相对于 谷胱甘肽的保护作用。 我们将评估这些假设， 性质的差异可归因于结构差异 取代呋喃的反应性亲电代谢物之间。 初步的串联质谱研究的代谢2- 呋喃酰胺已经表明，主要的谷胱甘肽代谢物中， RAT掺入未改性的取代呋喃。 的表征 与蛋白质缀合的外源性衍生部分补充了 谷胱甘肽结合物的鉴定，因为 谷胱甘肽结合物进一步代谢为反应性亲电性 物种 串联质谱法将应用于 水解成单氨基后蛋白质缀合物的表征 酸水平。 随后的工作将探讨表征的水平， 完整蛋白质或衍生的寡肽。 建议的意义 研究源于先进的分析战略的发展， 一般重要性的研究异生物质代谢，连同 说明异生物质产生的结构/毒性关系 反应性亲电代谢物。

Many xenobiotics are metabolized by formation of reactive electrophilic intermediates and subsequent conjugation with glutathione, yielding readily excreted products. This detoxification route may be compromised if conjugation to glutathione is inefficient or if the glutathione conjugates are themselves metabolized to electrophilic products. In these instances, toxic effects may result from binding of xenobiotics to cellular protein. Characterization of glutathione and protein conjugate metabolites can provide information on the structure of electrophilic intermediates, facilitating the understanding of structure-toxicity relationships. This is a particularly demanding analytical problem because formation of the electrophilic species may represent a quantitatively minor metabolic route and because structural characterization must be conducted in the presence of an excess of other components coextracted from the biological sample. We have developed a strategy for the screening and characterization of glutathione conjugates based on the use of stable isotope labelling and multiple scan modes in fast atom bombardment/tandem mass spectrometry (MS/MS and MS/MS/MS). This approach permits the direct analysis of complex biological extracts, with minimal sample preparation and correspondingly low risk of failure to observe metabolites of interest. Coupled liquid chromatography-electrospray ionization/tandem mass spectrometry provides a complementary approach of particular value for the recognition and characterization of quantitatively minor glutathione conjugate metabolites. These techniques will be applied to the analysis of glutathione conjugates (formed in vivo and in vitro) of several substituted furans, a class of compounds which shows marked toxicological variability with respect to the protective effect of glutathione. We will assess the hypothesis that these differences in properties may be attributed to structural differences between the reactive electrophilic metabolites of the substituted furans. Preliminary tandem mass spectrometric studies of the metabolism of 2- furamide have indicated that the principal glutathione metabolite in the rat incorporates the unmodified substituted furan. The characterization of xenobiotic-derived moieties conjugated to protein supplements the identification of glutathione conjugates because of the possibility of further metabolism of glutathione conjugates to reactive electrophilic species. Tandem mass spectrometric methods will be applied to the characterization of protein conjugates after hydrolysis to the single amino acid level. Subsequent work will explore characterization at the levels of intact protein or derived oligopeptides. The significance of the proposed research derives from the development of advanced analytical strategies of general importance to studies of xenobiotic metabolism, together with the elucidation of structure/toxicity relationships among xenobiotics yielding reactive electrophilic metabolites.