Open AccessEfficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase
Author(s) -
Chayan Bhattacharya,
Aninda Sundar Dey,
Navid J. Ayon,
William G. Gutheil,
Mridul Mukherji
Publication year - 2018
Publication title -
journal of visualized experiments
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.596
H-Index - 91
ISSN - 1940-087X
DOI - 10.3791/57798
Subject(s) - dna demethylation , 5 hydroxymethylcytosine , epigenetics , demethylation , chromosomal translocation , 5 methylcytosine , dioxygenase , methyltransferase , dna , dna glycosylase , chemistry , mutant , biology , tandem mass spectrometry , dna methylation , gene , biochemistry , microbiology and biotechnology , methylation , dna repair , mass spectrometry , chromatography , gene expression
The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair. Inactivation of the TET2 gene due to genetic mutations or by other epigenetic mechanisms is associated with a poor prognosis in patients with diverse cancers, especially hematopoietic malignancies. Here, we describe an efficient single step purification of enzymatically active untagged human TET2 dioxygenase using cation exchange chromatography. We further provide a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach that can separate and quantify the four normal DNA bases (A, T, G, and C), as well as the four modified cytosine bases (5-methyl, 5-hydroxymethyl, 5-formyl, and 5-carboxyl). This assay can be used to evaluate the activity of wild type and mutant TET2 dioxygenases.