A High-Throughput Mass Spectrometry Assay Coupled with Redox Activity Testing Reduces Artifacts and False Positives in Lysine Demethylase Screening

In this report, we demonstrate the utility of self-assembled monolayer desorption/ionization (SAMDI) mass spectrometry for the investigation of quantitative KDM enzyme kinetics and for high-throughput screening for KDM inhibitors. SAMDI can be performed in 384-well format and rapidly allows reaction components to be purified prior to injection into a mass spectrometer, without a throughput-limiting liquid chromatography step. We developed sensitive and robust assays for KDM1A (LSD1, AOF2) and KDM4C (JMJD2C, GASC1) and screened 13,824 compounds against each enzyme. Hits were rapidly triaged using a redox assay to identify compounds that interfered with the catalytic oxidation chemistry used by the KDMs for the demethylation reaction. We find that overall this high-throughput mass spectrometry platform coupled with the elimination of redox active compounds leads to a hit rate that is manageable for follow-up work.

Source: Journal of Biomolecular Screening - June 19, 2015 Category: Molecular Biology Authors: Wigle, T. J., Swinger, K. K., Campbell, J. E., Scholle, M. D., Sherrill, J., Admirand, E. A., Boriack-Sjodin, P. A., Kuntz, K. W., Chesworth, R., Moyer, M. P., Scott, M. P., Copeland, R. A. Tags: Original Research Source Type: research