A rapid mass spectrometric method for the measurement of catalytic activity of ten-eleven translocation enzymes.
A rapid mass spectrometric method for the measurement of catalytic activity of ten-eleven translocation enzymes.
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一种快速质谱法，用于测量十个时期易位酶的催化活性。
DOI：
10.1016/j.ab.2017.06.011
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发表时间：
2017-10-01
影响因子：
2.9
通讯作者：
Islam K
中科院分区：
文献类型：
--
作者：
Sudhamalla B;Dey D;Breski M;Islam K
Enzymatic methylation at carbon five on cytosine (5mC) in DNA is a hallmark of mammalian epigenetic programming and is critical to gene regulation during early embryonic development. It has recently been shown that dynamic erasure of 5mC by three members of the ten-eleven translocation (TET) family plays a key role in cellular differentiation. TET enzymes belong to Fe (II)- and 2-ketoglutarate (2KG) dependent dioxygenases that successively oxidize 5mC to 5-hydroxymethyl cytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5CaC), thus providing a chemical basis for the removal of 5mC which once was thought to be a permanent mark in mammalian genome. Since then a wide range of biochemical assays have been developed to characterize TET activity. Majority of these methods require multi-step processing to detect and quantify the TET-mediated oxidized products. In this study, we have developed a MALDI mass spectrometry based method that directly measures the TET activity with high sensitivity while eliminating the need for any intermediate processing steps. We applied this method to the measurement of enzymatic activity of TET1 and 3, Michaleis-Menten parameters (KM and kcat) of TET-2KG pairs and inhibitory concentration (IC50) of known small-molecule inhibitors of TETs. We further demonstrated the suitability of the assay to analyze chemoenzymatic labeling of 5hmC by β-glucosyltransferase, highlighting the potential for broad application of our method in deconvoluting the functions of novel DNA demethylases.
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影响因子：
13.8
作者：
Song, Chun-Xiao;He, Chuan
通讯作者：
He, Chuan
影响因子：
62.1
作者：
Lu X;Zhao BS;He C
通讯作者：
He C
影响因子：
4.2
作者：
Marholz, Laura J.;Wang, Wei;Zheng, Yu;Wang, Xiang
通讯作者：
Wang, Xiang
影响因子：
--
作者：
Liu MY;DeNizio JE;Kohli RM
通讯作者：
Kohli RM
影响因子：
2.7
作者：
Hamada, Shohei;Kim, Tae-Dong;Miyata, Naoki
通讯作者：
Miyata, Naoki