title: Sensitive and simultaneous determination of 5-methylcytosine and its oxidation products in genomic DNA by chemical derivatization coupled with liquid chromatography-tandem mass spectrometry analysis.
abstract: Cytosine methylation (5-methylcytosine, 5-mC) in genomic DNA is an important epigenetic mark that has regulatory roles in diverse biological processes. 5-mC can be oxidized stepwise by the ten-eleven translocation (TET) proteins to form 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC), and 5-carboxylcytosine (5-caC), which constitutes the active DNA demethylation pathway in mammals. Owing to the extremely limited contents of endogenous 5-mC oxidation products, no reported method can directly determine all these cytosine modifications simultaneously. In the current study, we developed selective derivatization of cytosine moieties with 2-bromo-1-(4-dimethylamino-phenyl)-ethanone (BDAPE) coupled with liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) for the simultaneous determination of these cytosine modifications in genomic DNA. The chemical derivatization notably improved the liquid chromatography separation and dramatically increased detection sensitivities of these cytosine modifications. The limits of detection (LODs) of the derivatives of 5-mC, 5-hmC, 5-foC, and 5-caC were 0.10, 0.06, 0.11, and 0.23 fmol, respectively. Using this method, we successfully quantified 5-mC, 5-hmC, 5-foC, and 5-caC in genomic DNA from human colorectal carcinoma (CRC) tissues and tumor-adjacent normal tissues. The results demonstrated significant depletion of 5-hmC, 5-foC, and 5-caC in tumor tissues compared to tumor-adjacent normal tissues, and the depletion of 5-hmC, 5-foC, and 5-caC may be a general feature of CRC; these cytosine modifications could serve as potential biomarkers for the early detection and prognosis of CRC. Moreover, the marked depletion of 5-hmC, 5-foC, and 5-caC may also have profound effects on epigenetic regulation in the development and formation of CRC.
authors: ∥Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan, Hubei 430072, P.R. China.|§Guangxi Zhuang Autonomous Region Center for Disease Prevention and Control, Nanning, Guangxi 530028, P.R. China.
authors: Yang
authors: Y
authors: Tang
authors: ∥Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan, Hubei 430072, P.R. China.
authors: Shu-Jian
authors: SJ
authors: Zheng
authors: ∥Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan, Hubei 430072, P.R. China.|‡Department of Pathology, Hubei Cancer Hospital, Wuhan, Hubei 430079, P.R. China.
authors: Chu-Bo
authors: CB
authors: Qi
authors: ∥Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan, Hubei 430072, P.R. China.
authors: Yu-Qi
authors: YQ
authors: Feng
authors: ∥Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan, Hubei 430072, P.R. China.
authors: Bi-Feng
authors: BF
authors: Yuan
mesh_terms: D044503:5-Methylcytosine / Q000032:analysis* / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods*; D015179:Colorectal Neoplasms / Q000473:pathology; D004247:DNA / Q000737:chemistry*; D019175:DNA Methylation; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D007659:Ketones / Q000737:chemistry; D057230:Limit of Detection; D010084:Oxidation-Reduction; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors
chemical_list: D007659:Ketones; D044503:5-Methylcytosine; D004247:DNA