Quantitative LC-MS/MS analysis of 5-hydroxymethyl-2'-deoxyuridine to monitor the biological activity of J-binding protein

Anal Biochem. 2020 Dec 1:610:113930. doi: 10.1016/j.ab.2020.113930. Epub 2020 Aug 29.

Abstract

Base J replaces 1% of thymine in most kinetoplastid flagellates, and is implicated in transcription regulation. Base J is synthesized in two steps: first, a thymine base in DNA is converted to 5-hydroxymethyluracil by J-binding proteins (JBP1, JBP2); secondly, a glucosyl transferase glycosylates the 5-hydroxymethyluracil to form base J. Here, we present a highly sensitive and selective LC-MS/MS method to quantify the in vitro JBP1 activity on synthetic oligonucleotide substrates. The method demonstrated successful to support biochemical studies of JBPs and can be used as a template for additional JBP activity studies or for inhibitor screening in the future.

Keywords: 5-hydroxymethyl-2′-deoxyuridine; Base J; JBP1/2; LC-MS/MS.

MeSH terms

  • Chromatography, High Pressure Liquid*
  • DNA-Binding Proteins / metabolism*
  • Leishmania / metabolism
  • Protozoan Proteins / metabolism*
  • Substrate Specificity
  • Tandem Mass Spectrometry*
  • Thymidine / analogs & derivatives*
  • Thymidine / analysis
  • Thymidine / chemistry
  • Thymidine / metabolism

Substances

  • DNA-Binding Proteins
  • J-specific DNA-binding protein, protozoa
  • Protozoan Proteins
  • 5-hydroxymethyl-2'-deoxyuridine
  • Thymidine