不同水解方法对DNA加合物分析鉴定的影响及应用研究  

Comparison of Different Hydrolysis Methods in DNA Adducts Analysis and Application

作　　者：武海江 张雅姣 刘勤 陈佳 徐斌 徐华 谢剑炜 WU Hai-Jiang;ZHANG Ya-Jiao;LIU Qin;CHEN Jia;XU Bin;XU Hua;XIE Jian-Wei(State Key Laboratory of Toxicology and Medical Countermeasures,Institute of Pharmacology and Toxicology,Academy of Military Medical Sciences,Beijing 100850,China)

机构地区：[1]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

出　　处：《分析化学》2022年第7期1048-1056,I0009,共10页Chinese Journal of Analytical Chemistry

基　　金：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。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.

关 键 词：DNA水解 DNA加合物 液相色谱-质谱联用技术 

分 类 号：Q503[生物学—生物化学]