Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

J Vis Exp. 2018 Oct 15:(140):57798. doi: 10.3791/57798.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Video-Audio Media

MeSH terms

  • 5-Methylcytosine / analysis
  • 5-Methylcytosine / metabolism
  • Chromatography, Liquid*
  • Cytosine / analogs & derivatives
  • Cytosine / analysis
  • DNA / chemistry
  • DNA Demethylation
  • DNA-Binding Proteins / isolation & purification*
  • DNA-Binding Proteins / metabolism*
  • Dioxygenases
  • Enzyme Assays / methods*
  • Humans
  • Proto-Oncogene Proteins / isolation & purification*
  • Proto-Oncogene Proteins / metabolism*
  • Tandem Mass Spectrometry*

Substances

  • DNA-Binding Proteins
  • Proto-Oncogene Proteins
  • 5-Methylcytosine
  • Cytosine
  • DNA
  • Dioxygenases
  • TET2 protein, human