Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, PR China.
Anal Chem. 2013 Jun 18;85(12):6129-35. doi: 10.1021/ac4010869. Epub 2013 May 28.
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.
5- 甲基胞嘧啶（5-mC）是一种参与发育的重要表观遗传修饰，可以通过酶促转化为 5-羟甲基胞嘧啶（5-hmC）。5-hmC 被认为是活跃的 DNA 胞嘧啶去甲基化的中间产物，并且自身充当表观遗传标记。大多数哺乳动物细胞中的 5-hmC 含量较低，通过液相色谱-质谱联用（LC-MS）定量 5-hmC 时经常会受到未修饰核苷存在的离子抑制。为了解决这个问题，我们开发了一种方法，通过使用 T4 β-葡萄糖基转移酶将葡萄糖基选择性地转移到 5-hmC 的羟甲基部分，形成更亲水的残基（β-葡萄糖基-5-羟甲基-2'-脱氧胞苷，5-gmdC）。更亲水的 5-gmdC 可以通过 NH2-硅胶在液相色谱-串联质谱（LC-MS/MS）分析之前通过亲水相互作用选择性地富集，这消除了离子抑制并显著提高了检测灵敏度和准确性。使用这种方法，我们成功地定量了三种人类细胞系和七种酵母菌株的基因组 DNA 中的 5-hmC 含量。据我们所知，这是关于酵母模型生物中存在 5-hmC 的第一个报道。此外，两种裂殖酵母（Schizosaccharomyces pombe）酵母菌株中的 5-hmC 含量甚至高于 5-mC，表明 5-hmC 可能在酵母的生理功能中发挥重要作用。
