咪唑类离子液衍生化DNA的质谱碎裂机理及其在RUNX3基因表观遗传修饰位点检测的应用研究

The cytosine methylation in the promoter of RUNX3 gene inhibits its antineoplastic activity. Identification of these methylation sites can help the cancer treatment and prognosis. Mass-spectrometric technique has played an important role in biological macromolecules analysis such as DNA, peptide and protein. However, the detection of cytosine methylation and other epigenetic modification sites in RUNX3 by mass spectrometry analysis still faces challenges. One of the most challenges is the lack of fragment ions severely restricting to obtain the structural information of targets. Ionic liquid is a type of ion form compound consisting of organic cation and organic/inorganic anion. It has the high ionization efficiency in the ion source of mass spectrometer. Recently the imidazolyl ionic liquid has been investigated as derivatization reagent to promote the protein occurring in-source decay in matrix assistant laser dissociation ionization (MALDI) mass spectrometer. This in-source decay technique has applied in the detection of the protein mutation sites. In this project, imidazolyl ionic liquids are introduced in the DNA derivatization. Derived DNA targets are analyzed by means of MALDI in-source decay and electrospray ionization tandem mass spectrometry (ESI-MSn). The fragmentation mechanisms of the DNA and the roles of the imidazolyl ionic liquids are elucidated. Moreover, fragmentation rules and characteristic fragment ions are summarized to establish analytical method to detect the epigenetic modification sites of RUNX3 gene such as 5-methylcytosine (5-mC), 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC), 5-carboxylcytosine (5-acC). Achievement of this project can lay the foundation of exploring the reparing mechanism of RUNX3. Additionally, it can help to develop new tumor drug targets.

RUNX3基因的启动子胞嘧啶甲基化抑制其抗肿瘤功能。确定甲基化位点有助于肿瘤治疗及预后。质谱技术已广泛应用于DNA等生物大分子的分析中，而利用质谱技术检测RUNX3基因甲基化等表观遗传修饰位点仍存在困难，无法得到丰富的碎片离子是主要问题之一。离子液体是由有机阳离子和有机或无机阴离子组成的离子形化合物，在质谱中电离效率较高。近期研究发现利用咪唑类离子液对蛋白分子衍生化可促进其在基质辅助激光解析质谱（MALDI）中发生源内裂解。此方法已成功应用于蛋白质突变位点的检测。因此本项目拟利用离子液对DNA分子衍生化，研究其在MALDI质谱中的源内裂解和电喷雾质谱中的多级碎裂机理。并在此基础上，建立和发展检测RUNX3基因胞嘧啶甲基化（5-mC）、羟甲基化（5-hmC）、醛基化（5-foC）及羧基化（5-acC）位点的质谱分析方法。为探索RUNX3基因的甲基化修复机理奠定基础，并帮助开发肿瘤药物新靶点。

DNA与RNA的表观遗传修饰已证明与多种疾病密切相关。对此类修饰进行准确定性定量分析，有助于研究其与疾病发生、发展的作用机制，为临床分子诊断提供基础。根据质谱技术高灵敏度、高分析效率等优势，本项目提出了以液质联用技术为基础，结合多种样品处理手段，对临床样本中DNA与RNA的表观遗传修饰核苷进行定性定量分析，探索其与疾病发病的关系。本项目按照计划开展了以下研究工作：利用二甲胺基苯二甲酸酐作为衍生化试剂，开发5-甲基脱氧胞苷（5-mdC）、5-羟甲基脱氧胞苷（5hmdC）、5-醛基脱氧胞苷（5-fodC）和5-羧基脱氧胞苷（5-cadC）柱前衍生化联合液质联用分析策略，高灵敏的检测乳腺癌组织中这四种DNA甲基化修饰的含量，并发现这四种修饰物含量在不同乳腺癌分型中存在差异；利用聚离子液磁性纳米材料，针对乳腺癌患者尿液中游离代谢的5-hmdC、5-fodC和5-cadC进行特异性富集，并结合液质联用技术进行检测，首次在尿液样本中检测到5-fodC和5-cadC，并发现在乳腺癌患者尿液中这三种游离修饰核苷存在明显变化；利用琥珀酸作为流动相添加剂，开发液质联用技术检测糖尿病与糖尿病合并肾病患者血清中多种表观遗传修饰的游离核苷检测方法，发现6-甲基腺苷（m6A）、肌苷（I）和假尿嘧啶（φ）含量在不同疾病组间发生动态变化；进一步以m6A为研究对象，利用细胞与动物模型探索了其在糖尿病肾病发病阶段调控SOCS/JAK/STAT通路的作用机制。本项目的开展丰富了质谱分析对DNA与RNA表观修饰核苷的检测技术，同时帮助临床潜在生物标志物的开发，对乳腺癌、糖尿病肾病的早期诊断和治疗具有较为重要的理论意义与应用价值。

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