Accessing Protein Methyltransferase and Demethylase Enzymology Using Microfluidic Capillary Electrophoresis

SummaryThe 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.

Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (170 K)Download as PowerPoint slideHighlights► Novel assay developed for lysine methyltransferases and demethylases ► Assay used to provide detailed insight into enzyme kinetics ► Demonstration of ability to profile small molecules inhibitors ► Mechanism of action of G9a inhibitors probed