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.

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.

Chemical labeling coupled with LC-MS enables the sensitive and simultaneous detection of the oxidative products of 5-methylcytosine. With this method, we can determine 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine in RNA of mammals.

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