Investigation of the metabolic changes caused by isocitrate dehydrogenase mutations using mass spectrometry

以前已经发现异柠檬酸脱氢酶突变与肿瘤细胞生长相关，特别是精氨酸132突变为组氨酸（R132 H），尽管已经观察到其他突变。这些突变在大多数2级和3级人类胶质瘤中发现，特别是影响20至40岁之间的患者，目前是所有癌症类型中5年生存率最低的癌症之一，使其成为主要的临床靶点。R132 H突变破坏了异柠檬酸向2-酮戊二酸的正常柠檬酸循环转化，并催化两种物种产生2-羟基戊二酸，这可能是由于与异柠檬酸脱氢酶活性位点的竞争性结合，这已被证明是由R132调节的。目前尚不清楚2-羟基戊二酸上调的生理学目的，需要进一步研究，因为已经假设了几种竞争性作用机制。这些包括2-酮戊二酸依赖性加氧酶的抑制，其改变参与细胞分化和蛋白质翻译后修饰的基因的表达。然而，在早期胶质瘤发展中观察到该突变，表明其具有促癌作用。在其他癌症类型中也观察到功能缺失突变，这导致富马酸盐和琥珀酸盐的积累，这也是主要关注的柠檬酸循环代谢物。质谱法仍然是代谢组学的主要分析方法，由于其高灵敏度和分辨率，可以检测飞摩尔浓度的代谢物离子。这允许鉴定作为细胞过程产物的小分子。代谢组学的最新进展，特别是离子色谱与质谱联用，允许小的极性和生理离子代谢物，这是没有观察到使用正常的色谱技术，被确定在复杂的mixture.This工作的目的是提高方法的定量和定性分析代谢物的电喷雾电离质谱的范围内的生物系统。野生型和突变型细胞系以及各种组织的代谢物表征。这将包括优化离子色谱法和其他色谱技术，以便通过数据库比较和定量来分离和鉴定代谢物，从而测量细胞浓度。将需要异柠檬酸脱氢酶突变的各种细胞系的组织培养，并使用稳定同位素标记的培养基通过碳通量分析来跟踪糖酵解和柠檬酸循环途径，最终目的是确定药物干预的靶点，以破坏癌细胞的生长。该项目福尔斯EPSRC物理科学-分析科学研究领域。

Isocitrate dehydrogenase mutations have previously been found to be associated with tumour cell growth, especially the mutation of arginine 132 to histidine (R132H) although other mutations have been observed. These mutations are found in the majority of grade 2 and grade 3 gliomas in humans, especially impacting those between the ages of 20 and 40 and currently have one of the lowest 5-year survival rates of all cancer types making them a prime clinical target. The R132H mutation disrupts the normal citric acid cycle conversion of isocitrate to 2-oxoglutarate and catalyses the production of 2-hydroxyglutarate from both species, possibly due to competitive binding to the isocitrate dehydrogenase active site which has been shown to be moderated by R132. The physiological purpose for the upregulation of 2-hydroxyglutarate is not currently known and requires further investigation as several competing mechanisms of action have been postulated. These include the inhibition of 2-oxoglutarate dependent oxygenases, which alters the expression of the genes involved in cell differentiation and posttranslational modification of proteins. The mutation has however, been observed in early glioma development suggesting a pro-oncogenic role. Loss of function mutations have also been observed in other cancer types which results in the accumulation of fumarate and succinate which are also citric acid cycle metabolites of prime interest.Mass spectrometry remains the primary analytical method for metabolomics due to its high sensitivity and resolution which allow the detection of metabolite ions at femtomolar concentrations. This allows the identification of small molecules that are the products of cellular processes. Recent advances in metabolomics, especially ion-chromatography coupled with mass spectrometry, allow small polar and physiologically ionic metabolites, which are not observed using normal chromatographic techniques, to be identified in complex mixtures.This work aims to improve the methods of quantitative and qualitative analysis of metabolites by electrospray ionisation mass spectrometry of a range of biological systems. The characterisation of metabolites from wild-type and mutant cell lines and various tissues. This will include the optimisation of ion chromatography and other chromatographic techniques to allow the separation and identification of metabolites with database comparison and quantification to measure cellular concentrations. Tissue culture of various cell lines with isocitrate dehydrogenase mutations will be required and the use of stable isotope labelled media will be used to follow glycolytic and citric acid cycle pathways by carbon flux analysis with the eventual aim of the identification of targets for pharmaceutical intervention to disrupt cancer cell growth.This project falls within the EPSRC Physical Sciences - Analytical Science research area.