Vertical Ultrafiltration-Facilitated DNA Digestion for Rapid and Sensitive UHPLC-MS/MS Detection of DNA Modifications

CORC > 生态环境研究中心 > 中国科学院生态环境研究中心 > 环境化学与生态毒理学国家重点实验室

	Vertical Ultrafiltration-Facilitated DNA Digestion for Rapid and Sensitive UHPLC-MS/MS Detection of DNA Modifications
	Lai, Weiyi; Lyu, Cong; Wang, Hailin
刊名	ANALYTICAL CHEMISTRY
	2018-06-05
卷号	90 期号:11 页码:6859-6866
ISSN号	0003-2700
文献子类	Article
英文摘要	LC-MS/MS technologies provide important and powerful analytical tools for chemical structure-dependent identification and quantification of epigenetically crucial DNA modifications. To perform LC-MS/MS analysis, it is better to convert DNA to 2'-deoxynucleosides through enzymatic digestion. Here, we observed that inorganic cations Na+ and K+ and phosphate buffers, which were often found in various DNA solutions, significantly inhibited DNA digestion as catalyzed by typical set of DNase I, snake venom phosphodiesterase, and calf intestine alkaline phosphatase, leading to poor or varying performance on UHPLC-MS/MS analysis. We then developed an efficient and unique vertical-ultrafiltration approach, enabling us to remove these inorganic salts without DNA loss. Consequently, the removal of inorganic salts by ultrafiltration facilitated the followed DNA digestion and thus enhanced the final UHPLC-MS/MS detection. Benefiting from the developed vertical-ultrafiltration approach, it is also feasible to integrate the desalting step with the other two steps of DNA digestion and protein removal. By investigating the time course of DNA digestion, we observed a differential release rate of 2'-deoxycytidine and 5-methyl-2'-deoxycytidine causing a measurement bias on the methylation frequency. We further exploited Mg2+ to eliminate this bias by stimulating DNase set-based DNA digestion. These innovative approaches enable us to perform rapid, sensitive, and robust UHPLC-MS/MS analysis of methylated DNA 2'-deoxycytidine, demethylation intermediates, and probably other DNA modifications.
内容类型	期刊论文
源URL	[http://ir.rcees.ac.cn/handle/311016/41081]
专题	生态环境研究中心_环境化学与生态毒理学国家重点实验室
推荐引用方式 GB/T 7714	Lai, Weiyi,Lyu, Cong,Wang, Hailin. Vertical Ultrafiltration-Facilitated DNA Digestion for Rapid and Sensitive UHPLC-MS/MS Detection of DNA Modifications[J]. ANALYTICAL CHEMISTRY,2018,90(11):6859-6866.
APA	Lai, Weiyi,Lyu, Cong,&Wang, Hailin.(2018).Vertical Ultrafiltration-Facilitated DNA Digestion for Rapid and Sensitive UHPLC-MS/MS Detection of DNA Modifications.ANALYTICAL CHEMISTRY,90(11),6859-6866.
MLA	Lai, Weiyi,et al."Vertical Ultrafiltration-Facilitated DNA Digestion for Rapid and Sensitive UHPLC-MS/MS Detection of DNA Modifications".ANALYTICAL CHEMISTRY 90.11(2018):6859-6866.