期刊
CHEMISTRY-A EUROPEAN JOURNAL
卷 19, 期 3, 页码 1035-1041出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/chem.201203129
关键词
liquid chromatography; 5-methylcytosine; mixed-mode retention; nucleobases; polymers
资金
- National Basic Research Program of China (973 Program) [2012CB720600, 2012CB720601]
- National Natural Science Foundation of China [91017013, 31070327, 21205091]
- Natural Science Fund for Creative Research Groups, NSFC [20921062]
- Fundamental Research Funds for the Central Universities
- Natural Science Foundation of Hubei Province [2011CDB440]
A hyper-cross-linked polymer monolithic column, poly(methacrylatoethyl trimethyl ammonium-co-vinylbenzene chloride-co-divinylbenzene) (MATE-co-VBC-co-DVB) with phenyl and quaternary ammonium groups was successfully prepared in the current study. The prepared monolith possesses large specific surface area, narrow mesopore size distribution and high column efficiency. The poly(MATE-co-VBC-co-DVB) monolithic column was demonstrated to have strong anion exchange/reversed-phase (SAX/RP) mixed-mode retention for analytes on capillary liquid chromatography (cLC). By using this monolithic column, we developed a rapid and sensitive method for the detection of DNA methylation. Our results showed that six nucleobases (adenine, guanine, cytosine, thymine, uracil, and 5-methylcytosine (5-mC)) can be baseline separated within 15 min by electrostatic repulsion and hydrophobic interactions between nucleobases and the monolithic stationary phase. The limit of detection (LOD, signal/noise=3) of 5-mC is 0.014 pmol and endogenous 5-mC can be distinctly detected by using only 10 ng genomic DNA, which is comparable to that obtained by mass spectrometry analysis. Furthermore, by using the method developed here, we found that DNA methylation inhibitor 5-azacytidine (5-aza-C) and 5-aza-2'-deoxycytidine (5-aza-CdR) could induce a significant decrease of genome-wide DNA methylation in human lung carcinoma cells (A549) and cervical carcinoma cells (HeLa).
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