title: Determination of DNA and RNA Methylation in Circulating Tumor Cells by Mass Spectrometry.
abstract: DNA methylation (5-methylcytosine, 5-mC) is the best characterized epigenetic mark that has regulatory roles in diverse biological processes. Recent investigation of RNA modifications also raises the possible functions of RNA adenine and cytosine methylations on gene regulation in the form of "RNA epigenetics." Previous studies demonstrated global DNA hypomethylation in tumor tissues compared to healthy controls. However, DNA and RNA methylation in circulating tumor cells (CTCs) that are derived from tumors are still a mystery due to the lack of proper analytical methods. In this respect, here we established an effective CTCs capture system conjugated with a combined strategy of sample preparation for the captured CTCs lysis, nucleic acids digestion, and nucleosides extraction in one tube. The resulting nucleosides were then further analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). With the developed method, we are able to detect DNA and RNA methylation (5-methyl-2'-deoxycytidine, 5-methylcytidine, and N(6)-methyladenosine) in a single cell. We then further successfully determined DNA and RNA methylation in CTCs from lung cancer patients. Our results demonstrated, for the first time, a significant decrease of DNA methylation (5-methyl-2'-deoxycytidine) and increase of RNA adenine and cytosine methylations (N(6)-methyladenosine and 5-methylcytidine) in CTCs compared with whole blood cells. The discovery of DNA hypomethylation and RNA hypermethylation in CTCs in the current study together with previous reports of global DNA hypomethylation in tumor tissues suggest that nucleic acid modifications play important roles in the formation and development of cancer cells. This work constitutes the first step for the investigation of DNA and RNA methylation in CTCs, which may facilitate uncovering the metastasis mechanism of cancers in the future.
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University , Wuhan 430072, Peoples' Republic of China.
authors.forename: Wei
authors.initials: W
authors.lastname: Huang
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University , Wuhan 430072, Peoples' Republic of China.|Department of Pathology, Hubei Cancer Hospital , Wuhan, Hubei 430079, Peoples' Republic of China.
authors.forename: Chu-Bo
authors.initials: CB
authors.lastname: Qi
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University , Wuhan 430072, Peoples' Republic of China.
authors.forename: Song-Wei
authors.initials: SW
authors.lastname: Lv
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University , Wuhan 430072, Peoples' Republic of China.
authors.forename: Min
authors.initials: M
authors.lastname: Xie
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University , Wuhan 430072, Peoples' Republic of China.
authors.forename: Yu-Qi
authors.initials: YQ
authors.lastname: Feng
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University , Wuhan 430072, Peoples' Republic of China.
authors.forename: Wei-Hua
authors.initials: WH
authors.lastname: Huang
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University , Wuhan 430072, Peoples' Republic of China.
authors.forename: Bi-Feng
authors.initials: BF
authors.lastname: Yuan
mesh_terms: D002851:Chromatography, High Pressure Liquid; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000032:analysis* / Q000097:blood / Q000737:chemistry*; D006801:Humans; D008175:Lung Neoplasms / Q000097:blood / Q000737:chemistry* / Q000473:pathology; D061986:MCF-7 Cells; D009360:Neoplastic Cells, Circulating / Q000737:chemistry* / Q000473:pathology; D012334:RNA, Neoplasm / Q000032:analysis* / Q000097:blood / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
chemical_list: D004273:DNA, Neoplasm; D012334:RNA, Neoplasm