Determination of 5-hydroxymethyl-2-deoxycytidine in Rice by High-performance Liquid Chromatography-Tandem Mass Spectrometry with Isotope Dilution
Determination of 5-hydroxymethyl-2-deoxycytidine in Rice by High-performance Liquid Chromatography-Tandem Mass Spectrometry with Isotope Dilution
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同位素稀释高效液相色谱-串联质谱法测定大米中5-羟甲基-2-脱氧胞苷
DOI：
10.1080/00032719.2017.1286668
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发表时间：
2017-01-01
影响因子：
2
通讯作者：
Zhao, Rusong
中科院分区：
文献类型：
--
作者：
Wang, Xiaoli;Guo, Tao;Zhao, Rusong
5-羟甲基-2-脱氧胞苷（5-hmdC）在哺乳动物的表观遗传重构中起重要作用，最近在植物基因组中检测到痕量浓度。在这项研究中，5-hmdC的敏感性，特异性和快速评估的方法报告使用同位素稀释的高效液相色谱-串联质谱（HPLC-tandem MS）。该方法的回收率为75.6%~ 81.3%，日内和日间精密度均在5.81%以内。5-hmdC的定量限为5.0 pM。从水稻中提取基因组DNA，酶消化核苷酸，用于HPLC-串联MS测定。在盛稻18、盛香145和津稻263中，5-hmdC水平分别为1.32 +/- 0.08、1.64 +/- 0.12和1.98 +/- 0.07个修饰/百万核苷。应用HPLC-Tandem MS技术检测水稻基因组DNA中的5-hmdC，为进一步研究修饰胞嘧啶在水稻表观遗传调控中的作用奠定了基础。
5-hydroxymethyl-2-deoxycytidine (5-hmdC), which plays important roles in the epigenetic remodeling in mammals, has been recently determined in plant genomes at trace concentrations. In this study, a method for the sensitive, specific, and rapid assessment of 5-hmdC in rice is reported using high-performance liquid chromatography-tandem mass spectrometry (HPLC-tandem MS) with isotope dilution. The recovery of this method was from 75.6% to 81.3% for 5-hmdC and the intra- and inter-day precision values were within 5.81%. The quantification limit for 5-hmdC was 5.0pM. Genomic DNA from rice was extracted and enzymatically digested for nucleotides for the HPLC-tandem MS assay. The 5-hmdC levels were 1.32 +/- 0.08, 1.64 +/- 0.12, and 1.98 +/- 0.07 modifications per million nucleosides in the Shengdao-18, Shengxiang-145, and Jindao-263 rice cultivars. The application of HPLC-tandem MS for 5-hmdC demonstrated its presence in the genomic DNA of rice and is useful in further studying the role of modified cytosine in the epigenetic regulation of rice.