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

Abstract

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 tumoradjacent 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.

Quantification and mapping of DNA modifications

“…We and others previously utilized 2-bromo-1- benzoic anhydride to simultaneously label 5mC, 5hmC, 5fC, and 5caC in DNA, [98][99][100][101] which led up to 313 fold increase of detection sensitivities of DNA modifications. Similarly, some other chemical derivatization strategies were developed to enable the sensitive detection of DNA modifications, such as glycosylation of 5hmC by T4 β-glucosyltransferase, 102 derivatization of 5fC, 5caC and 5fU by Girard's reagents (Girard D, T, and P) [103][104][105] and hydrazine reagents (Me 2 N, Et 2 N, and i-Pr 2 N), 106 and derivatization of 5fC by dansylhydrazide (DNSH).…”

Transcriptomic and Epigenomic Landscape in Rett Syndrome

“…To the best of our knowledge, the genomic distribution of 5-formylcytosine (fC) and 5-carboxylcytosine (caC), deriving from the further oxidation of mC/hmC [78] in RTT remain largely unexplored. Aberrant levels of fC and caC have been described in some human diseases [79,80]. Therefore, it would be interesting to investigate them in RTT patients and/or in RTT mouse models to gain further insights into the relationship between MeCP2 and the regulation of DNA methylation and its derivatives.…”

Adenine derivatization for LC-MS/MS epigenetic DNA modifications studies on monocytic THP-1 cells exposed to reference particulate matter

“…For instance, those methods used derivatives containing a bromoacetone functional group, which specifically reacts with cytosine on the 4-N and 3-N position forming penta-cylic derivatives [15][16][17]. Guo et al used a different derivative containing an anhydride group, which reacts with the primary amine group on the 4-N position of cytosine [18]. After derivatization both the retention behavior of the derivatives during liquid chromatography and the ionization efficiency of derivatives in the MS-source were significantly improved.…”