Accessing protein methyltransferase and demethylase enzymology using microfluidic capillary electrophoresis

Tim J. Wigle, Laurel M. Provencher, Jacqueline L. Norris, Jian Jin, Peter J. Brown, Stephen V. Frye, William P. Janzen

Research output: Contribution to journalArticlepeer-review

41 Scopus citations

Abstract

The discovery of small molecules targeting the >80 enzymes that add (methyltransferases) or remove (demethylases) methyl marks from lysine and arginine residues, most notably present in histone tails, may yield unprecedented chemotherapeutic agents and facilitate regenerative medicine. To better enable chemical exploration of these proteins, we have developed a highly quantitative microfluidic capillary electrophoresis assay to enable full mechanistic studies of these enzymes and the kinetics of their inhibition. This technology separates small biomolecules, i.e., peptides, based on their charge-to-mass ratio. Methylation, however, does not alter the charge of peptide substrates. To overcome this limitation, we have employed a methylation-sensitive endoproteinase strategy to separate methylated from unmethylated peptides. The assay was validated on a lysine methyltransferase (G9a) and a lysine demethylase (LSD1) and was employed to investigate the inhibition of G9a by small molecules.

Original languageEnglish
Pages (from-to)695-704
Number of pages10
JournalChemistry and Biology
Volume17
Issue number7
DOIs
StatePublished - 2010
Externally publishedYes

Keywords

  • CHEMBIO
  • DNA

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