基于质谱技术的甲基化DNA氧化衍生物的高灵敏定量分析及其在癌症相关研究中的应用

项目名称： 基于质谱技术的甲基化DNA氧化衍生物的高灵敏定量分析及其在癌症相关研究中的应用

项目编号： No.21205091

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 分析化学

项目作者： 袁必锋

作者单位： 武汉大学

项目金额： 27万元

中文摘要： 最新发现的三种甲基化胞嘧啶（5-mC）氧化衍生物，即5-羟甲基胞嘧啶(5-hmC)、5-甲酰胞嘧啶(5-fC)和5-羧基胞嘧啶（5-caC），它们除了可能作为DNA去甲基化过程中的中间产物，还可为癌症研究和干细胞分化提供非常重要的信息。但是由于目前分析检测手段的限制，对体内甲基化DNA氧化衍生物（5-hmC、5-fC、5-caC）的精确定量分析方法仍然不够成熟。在本课题中，我们拟制备亲水性聚合物整体柱，建立基于质谱技术的甲基化DNA氧化衍生物高灵敏度、高精确度的在线富集-整体柱液相色谱-质谱分析的检测体系和气相色谱-质谱分析检测体系，实现一次性同时分离分析检测细胞中全部三种甲基化DNA氧化衍生物（5-hmC、5-fC、5-caC）。通过对甲基化DNA氧化衍生物的定量分析，为干细胞分化机制的阐明、癌症的风险评估、早期预警和诊断、抗癌药物新的作用通路的发现等提供新的实验分析方法。

中文关键词： DNA甲基化；甲基化DNA氧化衍生物；质谱分析；化学标记；癌症

英文摘要： Recent studies suggested that the three oxidized products of 5-methylcytosine (5-mC), 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) may be the intermediate products during DNA demethylation processing, which opens a new gate for understanding the intricate mechanism of DNA demethylation. In addition, the three oxidized products of 5-methylcytosine are also closely related to cancer formation and differentiation of stem cells. However, due to the limitation of current analytical methods, the quantitative detection of the in-vivo oxidized products of 5-methylcytosine, 5-hmC, 5-fC and 5-caC, is still a challenging task. And different research groups even obtained different results for the measurement of 5-hmC between stem cells and differentiated cells. To circumvent the problem, we are going to establish a highly sensitive, accurate and automated method for the analysis of 5-hmC, 5-fC and 5-caC by coupling hydrophilic monolithic column with ESI-QTOF mass spectrometry in current study. With this system, we can analyze 5-hmC, 5-fC and 5-caC at the same time. And by analyzing the change of the oxidized products of 5-methylcytosine in normal and cancer cells, it will provide a new method for the diagnosis and risk assessment of diseases. In addition, the proposal study will sh

英文关键词： DNA methylation；Oxidation products of 5-methylcytosine；Mass spectrometry analysis；Chemical labeling；Cancer