A rapid mass spectrometric method for the measurement of catalytic activity of ten-eleven translocation enzymes

- Active demethylation of 5mC by TET enzymes is one of the key mechanisms of epigenetic regulation in vertebrates.
- MALDI-based assay is developed to characterize catalytic activity of TET enzymes.
- The method is employed to determine kinetic parameters of TETs and IC50 of TET inhibitors.
- The assay is characterized by its minimum sample preparation, rapid and straightforward analysis.

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 TET2 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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