title: Hydrophilic material for the selective enrichment of 5-hydroxymethylcytosine and its liquid chromatography-tandem mass spectrometry detection.
abstract: 5-Methylcytosine (5-mC), an important epigenetic modification involved in development, can be converted enzymatically to 5-hydroxymethylcytosine (5-hmC). 5-hmC is considered an intermediate of active DNA cytosine demethylation and makes itself serve as an epigenetic mark. 5-hmC content in most mammalian cells is low and the quantification of 5-hmC by liquid chromatography-mass spectrometry (LC-MS) frequently suffers from ion suppression by the presence of unmodified nucleosides. To circumvent this problem, we developed a method to selectively transfer a glucosyl group to the hydroxymethyl moiety of 5-hmC and form a more hydrophilic residue (β-glucosyl-5-hydroxymethyl-2'-deoxycytidine, 5-gmdC) by using T4 β-glucosyltransferase. The more hydrophilic 5-gmdC can be selectively enriched by using NH2-silica via hydrophilic interaction prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, which eliminates the ion suppression and significantly improves the detection sensitivity and accuracy. Using this method, we successfully quantified 5-hmC content in genomic DNA of three human cell lines and seven yeast strains. To the best of our knowledge, this is the first report about the existence of 5-hmC in the model organism of yeast. In addition, the contents of 5-hmC in two yeast strains of Schizosaccharomyces pombe are even higher than those of 5-mC, indicating that 5-hmC may play important roles on the physiological functions of yeast.
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, PR China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Tang
authors.forename: Jie-Mei
authors.initials: JM
authors.lastname: Chu
authors.forename: Wei
authors.initials: W
authors.lastname: Huang
authors.forename: Jun
authors.initials: J
authors.lastname: Xiong
authors.forename: Xi-Wen
authors.initials: XW
authors.lastname: Xing
authors.forename: Xiang
authors.initials: X
authors.lastname: Zhou
authors.forename: Yu-Qi
authors.initials: YQ
authors.lastname: Feng
authors.forename: Bi-Feng
authors.initials: BF
authors.lastname: Yuan
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives; D002853:Chromatography, Liquid / Q000379:methods; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D019169:Jurkat Cells; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine