A validated quantitative liquid chromatography-tandem quadrupole mass spectrometry method for monitoring isotopologues to evaluate global modified cytosine ratios in genomic DNA
A validated quantitative liquid chromatography-tandem quadrupole mass spectrometry method for monitoring isotopologues to evaluate global modified cytosine ratios in genomic DNA
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DOI：
10.1016/j.jchromb.2014.01.050
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发表时间：
2014-03-15
影响因子：
3
通讯作者：
Tomioka, Yoshihisa
中科院分区：
文献类型：
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作者：
Tsuji, Makoto;Matsunaga, Hironori;Tomioka, Yoshihisa
5-羟甲基胞嘧啶（5 hmC）和5-甲基胞嘧啶（5 mC）代表对DNA的重要表观遗传修饰，并且需要灵敏的分析方法来确定肿瘤细胞或培养细胞系的基因组DNA中的5 hmC水平，因为5 hmC在这些细胞中以特别低的水平存在。我们已经开发了一种灵敏的液相色谱-串联四极杆质谱法定量5-羟甲基脱氧胞苷（5 hmdC），5-甲基脱氧胞苷（5 mdC），脱氧鸟苷（dG）水平使用稳定的同位素标记的内标物，并使用这种方法来估计从少量细胞制备的基因组DNA中的2个修饰的胞嘧啶的整体水平。5 hmdC、5 mdC和dG的定量限分别为20 pM、2 nM和10 nM。同位素体的MRM转换（同位素体-MRM）用于定量5 mdC和dG水平，因为这些核苷相对于5 hmdC的丰度。对于丰富的核苷使用同位素-MRM还可以避免检测器的饱和，并且允许同时分析所有三种核苷，而不需要将样品稀释和重新注入仪器中。在对基因组DNA水解产物中的每种核苷进行定量后，估计修饰的胞嘧啶核苷与dG的总体比率。用200 ng DNA估算5 hmC的下限小于0.001%，我们发现急性髓系白血病的融合蛋白MLL-TET 1不产生5 hmC，但能干扰TET 1的活性，使其在细胞内产生5 hmC。因此，我们的分析方法是一个有价值的工具，进一步的研究，旨在更深入地了解修饰的胞嘧啶在细胞的表观遗传调控的作用。(C)2014爱思唯尔有限公司版权所有。
5-Hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) represent important epigenetic modifications to DNA, and a sensitive analytical method is required to determine the levels of 5hmC in the genomic DNA of tumor cells or cultured cell lines because 5hmC is present at particular low levels in these cells. We have developed a sensitive liquid chromatography-tandem quadrupole mass spectrometric method for quantifying 5-hydroxymethyldeoxycytidine (5hmdC), 5-methyldeoxycytidine (5mdC), and deoxyguanosine (dG) levels using stable isotope labeled internal standards, and used this method to estimate the global level of 2 modified cytosines in genomic DNA prepared from small number of cells. The quantification limits for 5hmdC, 5mdC and dG were 20 pM, 2 nM and 10 nM, respectively. MRM transitions for isotopologue (isotopologue-MRM) were used to quantify the 5mdC and dG levels because of the abundance of these nucleosides relative to 5hmdC. The use of isotopologue-MRM for the abundant nucleosides could also avoid the saturation of the detector, and allow for all three nucleosides to be analyzed simultaneously without the need for the dilution and re-injection of samples into the instrument. The global ratios of modified cytosine nucleosides to dG were estimated following the quantification of each nucleoside in the hydrolysate of genomic DNA. The limit of estimation for the global 5hmC level was less than 0.001% using 200 ng of DNA.Using this method, we found that MLL-TET1, which a fusion protein in acute myelogenous leukemia, did not produce 5hmC, but interfered with TET1 activity to produce 5hmC in cells. Our analytical method is therefore a valuable tool for further studies aiming at a deeper understanding of the role of modified cytosine in the epigenetic regulation of cells. (C) 2014 Elsevier B.V. All rights reserved.