A sensitive mass spectrometry method for simultaneous quantification of DNA methylation and hydroxymethylation levels in biological samples.
A sensitive mass spectrometry method for simultaneous quantification of DNA methylation and hydroxymethylation levels in biological samples.
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一种敏感的质谱法，用于同时定量生物样品中的DNA甲基化和羟甲基水平。
DOI：
10.1016/j.ab.2011.01.026
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发表时间：
2011-05-15
影响因子：
2.9
通讯作者：
Faull KF
中科院分区：
文献类型：
--
作者：
Le T;Kim KP;Fan G;Faull KF
最近在胚胎干细胞和有丝分裂后神经元中发现了5-羟甲基胞嘧啶(5HmC)，这引发了对同一样本中5-甲基胞嘧啶(5mC)和5HmC的定量测量的需要。我们建立了一种用LC-ESI-MS/MS-MRM同时测定消化后基因组DNA中5mC和5HmC的方法。该方法快速、可靠、准确，比目前薄层层析末端标记法、糖基化核素标记法等5HmC定量方法更灵敏。只需要50 ng消化的基因组DNA就可以检测到小鼠胚胎干细胞DNA中存在0.1%的5hmC。使用这一程序，我们发现人诱导的多能干细胞与亲代成纤维细胞相比，5mC和5hmC水平显著增加，这表明细胞重新编程过程中DNA甲基化和羟甲基化的动态调节。
The recent discovery of 5-hydroxymethyl-cytosine (5hmC) in embryonic stem cells and post-mitotic neurons has triggered the need for quantitative measurements of both 5-methyl-cytosine (5mC) and 5hmC in the same sample. We have developed a method using liquid chromatography electrospray ionization tandem mass spectrometry with multiple reaction monitoring (LC-ESI-MS/MS-MRM) to simultaneously measure levels of 5mC and 5hmC in digested genomic DNA. This method is fast, robust and accurate, and is more sensitive than the current 5hmC quantitation methods such as end-labeling with thin-layer chromatography and radio-labeling by glycosylation. Only 50 ng of digested genomic DNA is required to measure the presence of 0.1% 5hmC in DNA from mouse embryonic stem cells. Using this procedure we show that human induced pluripotent stem cells exhibit a dramatic increase in 5mC and 5hmC levels compared to parental fibroblast cells, suggesting a dynamic regulation of DNA methylation and hydroxymethylation during cellular reprogramming.
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影响因子：
46.9
作者：
Deng, Jie;Shoemaker, Robert;Xie, Bin;Gore, Athurva;LeProust, Emily M.;Antosiewicz-Bourget, Jessica;Egli, Dieter;Maherali, Nimet;Park, In-Hyun;Yu, Junying;Daley, George Q.;Eggan, Kevin;Hochedlinger, Konrad;Thomson, James;Wang, Wei;Gao, Yuan;Zhang, Kun
通讯作者：
Zhang, Kun
影响因子：
48
作者：
Flusberg, Benjamin A.;Webster, Dale R.;Lee, Jessica H.;Travers, Kevin J.;Olivares, Eric C.;Clark, Tyson A.;Korlach, Jonas;Turner, Stephen W.
通讯作者：
Turner, Stephen W.
Dnmt1 和 Dnmt3a 维持 DNA 甲基化并调节成人前脑神经元的突触功能
影响因子：
25
作者：
Feng, Jian;Zhou, Yu;Campbell, Susan L.;Le, Thuc;Li, En;Sweatt, J. David;Silva, Alcino J.;Fan, Guoping
通讯作者：
Fan, Guoping
影响因子：
4.6
作者：
Fan, GP;Martinowich, K;Sun, YE
通讯作者：
Sun, YE
影响因子：
5.2
作者：
Pick, Marjorie;Stelzer, Yonatan;Benvenisty, Nissim
通讯作者：
Benvenisty, Nissim