不同水解方法对DNA加合物分析鉴定的影响及应用研究

引用本文: 武海江, 张雅姣, 刘勤, 陈佳, 徐斌, 徐华, 谢剑炜. 不同水解方法对DNA加合物分析鉴定的影响及应用研究[J]. 分析化学, 2022, 50(7): 1048-1056. doi: 10.19756/j.issn.0253-3820.221018 shu

Citation: WU Hai-Jiang, ZHANG Ya-Jiao, LIU Qin, CHEN Jia, XU Bin, XU Hua, XIE Jian-Wei. Comparison of Different Hydrolysis Methods in DNA Adducts Analysis and Application[J]. Chinese Journal of Analytical Chemistry, 2022, 50(7): 1048-1056. doi: 10.19756/j.issn.0253-3820.221018 shu

不同水解方法对DNA加合物分析鉴定的影响及应用研究

军事医学研究院毒物药物研究所, 抗毒药物与毒理学国家重点实验室, 北京 100850

通讯作者: 谢剑炜,E-mail:xiejianwei@bmi.ac.cn

基金项目:
国家自然科学基金项目(No.22104153)资助。

摘要: DNA加合物的分析鉴定是研究DNA损伤的重要技术手段。采用不同的DNA水解方法可获得不同的碱基、核苷和核酸片段等产物,这些方法各具优势,可用于不同的分析目的。本研究以经典的烷基化试剂芥子气为模型化合物,进行溶液孵育与动物染毒实验。采用3种常用的水解方式(加热水解、酸水解和酶解)处理目标分析物,利用质谱技术对产物进行筛查与定量分析,系统地比较了这3种常用的水解方法对不同分析样本和具有不同加合形式的加合物的水解效率的影响。结果表明,不同修饰位点的DNA加合物的稳定性存在差异,鸟嘌呤与腺嘌呤的N位点加合物的结构较稳定,鸟嘌呤的O6位点加合物则对酸和热不稳定。在3种水解方式中,加热水解法的水解效率较低;酸水解法高效稳定,其产率较加热水解法提高了10.4%~94.9%,产物中碱基形式占比＞99%,适用于已知结构的DNA加合物的高通量、高灵敏质谱分析;酶解法可产生碱基与核苷等多种产物,产物信息丰富,适用于新结构以及未知结构加合物的筛查与鉴定,但因其酶解效率受DNA加合位点、基质效应与酶解条件等因素限制,定量分析加合物时其准确性受到影响。

关键词:

English

Comparison of Different Hydrolysis Methods in DNA Adducts Analysis and Application

State Key Laboratory of Toxicology and Medical Countermeasures, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China

Corresponding author: XIE Jian-Wei, xiejianwei@bmi.ac.cn

Received Date: 12 January 2022
Revised Date: 30 March 2022
Fund Project:
Supported by the National Natural Science Foundation of China (No.22104153).

Abstract: The identification and quantification of DNA adducts is an important technical issue in the research of DNA damage, epigenetics and genotoxic impurities screening. Various DNA hydrolysis methods with individual advantages are available for different applicable analytical purposes to produce bases, nucleosides or nucleic acid fragments. By taking the classic alkylation reagent mustard gas as a model compound, three common DNA hydrolysis methods including thermal hydrolysis, acid hydrolysis and enzymatic hydrolysis were comparatively used to pretreat the sample from in vitro incubation solution or in vivo animal exposure experiments. The produced DNA adducts were screened and quantitatively detected by mass spectrometry. Hydrolysis efficiency of DNA adducts in different analytical samples as well as different sites and adduct forms were investigated. Results showed that there were differences in the stability of DNA adducts derived from different base modification sites. The N-site adducts of guanine and adenine were relatively stable under each hydrolysis condition, while O6 site adducts of guanine were unstable to acid and thermal hydrolysis. In comparison, the efficiency of thermal hydrolysis was lower, while the acid hydrolysis was more efficient which improved 10.4%-94.9% hydrolysis yields and DNA-base adducts were more than 99% among the products, thus suitable for the quantitative analysis of structure-known DNA adducts by mass spectrometry. For enzymatic hydrolysis method, it could produce various adduct forms, such as base- and/or nucleoside-adducts, i.e., the product information was rich, which was suitable for screening and identification of structure-unknown adducts. However, the enzymatic hydrolysis efficiency was often affected by the factors such as DNA modification site, matrix effect and enzymatic hydrolysis conditions which might consequently affect the accuracy of DNA adduct quantification.

Key words:

DNA hydrolysis
/ DNA adducts
/ Liquid chromatography-tandem mass spectrometry

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沈阳化工大学材料科学与工程学院沈阳 110142

不同水解方法对DNA加合物分析鉴定的影响及应用研究

通讯作者: 谢剑炜,E-mail:xiejianwei@bmi.ac.cn

English

Comparison of Different Hydrolysis Methods in DNA Adducts Analysis and Application

Corresponding author: XIE Jian-Wei, xiejianwei@bmi.ac.cn

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通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

不同水解方法对DNA加合物分析鉴定的影响及应用研究

通讯作者: 谢剑炜,E-mail:xiejianwei@bmi.ac.cn

English

Comparison of Different Hydrolysis Methods in DNA Adducts Analysis and Application

Corresponding author: XIE Jian-Wei, xiejianwei@bmi.ac.cn

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

CCS Chemistry：颜徐州、鲍哲南： 你的皮肤可能是一片SPMs

CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

CCS Chemistry：静电组装导向聚合- 聚电解质纳米凝胶量化制备新策略

CCS Chemistry：金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的？

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