Mass spectrometry (MS)-based high-throughput screening (HTS) has tremendous potential as an alternative to current screening methods due to its speed, sensitivity, reproducibility and label-free readout. We recently reported that a new generation matrix-assisted laser desorption/ionization triple quadrupole (MALDI-QqQ) mass spectrometer is ideally suited for a variety of enzyme assays and screening protocols. However, all the targets measured to date had peptide substrates that were easily monitored by selected ion monitoring (SIM) without interference from the MALDI matrix. To further extend the application to enzymes with small molecule, non-peptide substrates, we evaluated this method for measuring enzyme activity and inhibition of acetylcholinesterase (AChE). Due to the potential of MALDI matrix interference, multiple reaction monitoring (MRM) was investigated for selective MS/MS transitions and to accurately measure the conversion of acetylcholine into choline. Importantly, ionization, detection and MRM transition efficiency differences between the substrate and product can be overcome by pre-balancing the MRM transitions during method development, thus allowing for a direct readout of the enzyme activity using the ratio of the substrate and product signals. Further validation of the assay showed accurate concentration-dependent inhibition measurements of AChE with several known inhibitors. Finally, a small library of 1008 drug-like compounds was screened at a single dose (10 microM) and the top 10 inhibitors from this primary screen were validated in a secondary screen to determine the rank order of inhibitory potency for each compound. Collectively, these data demonstrate that a MALDI-QqQMS-based readout platform is amenable to measuring small molecule substrates and products and offers significant advantages over current HTS methods in terms of speed, sensitivity, reproducibility and reagent costs.
Copyright 2009 John Wiley & Sons, Ltd.