Research Papers:

Accurate quantification of 5-Methylcytosine, 5-Hydroxymethylcytosine, 5-Formylcytosine, and 5-Carboxylcytosine in genomic DNA from breast cancer by chemical derivatization coupled with ultra performance liquid chromatography- electrospray quadrupole time of flight mass spectrometry analysis

Mengzhe Guo, Xiao Li, Liyan Zhang, Dantong Liu, Wencheng Du, Dengyang Yin, Nan Lyu, Guangyu Zhao, Cheng Guo and Daoquan Tang _

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Oncotarget. 2017; 8:91248-91257. https://doi.org/10.18632/oncotarget.20093

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Mengzhe Guo1,*, Xiao Li1,*, Liyan Zhang1,*, Dantong Liu1, Wencheng Du1, Dengyang Yin3, Nan Lyu4, Guangyu Zhao3, Cheng Guo2 and Daoquan Tang1

1Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical College, Xuzhou, Jiangsu 221004, China

2Cancer Institute, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China

3Jingjiang People’s Hospital, Jingjiang, Jiangsu 214500, China

4Xuzhou Central Hospital, Xuzhou, Jiangsu 221004, China

*These authors contributed equally to this work and are co-first authors

Correspondence to:

Daoquan Tang, email: [email protected]

Cheng Guo, email: [email protected]

Keywords: liquid chromatography mass spectrometry, DNA demethylation, breast cancer, derivatization, 5-methylcytosine

Received: June 22, 2017     Accepted: July 26, 2017     Published: August 09, 2017


The DNA demethylation pathway has been discovered to play a significant role in DNA epigenetics. This pathway removes the methyl group from cytosine, which is involved in the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) by ten-eleven translocation (TET) proteins. Then, 5-hmC can be iteratively oxidized to generate 5-formylcytosine (5-foC) and 5-carboxylcytosine (5-caC). However, 5-hmC, 5-foC, and 5-caC are hardly detected due to their low content. In this study, we have developed a LC-HRMS method coupled with derivatization to accurately and simultaneously quantify 5-mC levels, along with its oxidation products in genomic DNA. Derivatization was carried out using 4-dimethylamino benzoic anhydride, which has been shown to improve separation and enhance the detection sensitivity. Finally, we successfully applied this method towards the quantification of 5-mC, 5-hmC, 5-foC, and 5-caC in genomic DNA isolated from both human breast cancer tissue and tumor-adjacent normal tissue. We show that 5-foC and 5-caC are increased in tumor tissue. In addition, the levels of 5-mC, 5-hmC, 5-foC, and 5-caC measured in tumor tissue versus tumor-adjacent tissue were found to be distinct among different classifications. This suggests that cytosine modifiers could be used as potential biomarkers for determining the stage of development of breast cancer, as well as prognosis.

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