Rathore Rakesh, Corr Jay J, Lebre Daniel T, Seibel William L, Greis Kenneth D
Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH 45237, USA.
Rapid Commun Mass Spectrom. 2009 Oct 30;23(20):3293-300. doi: 10.1002/rcm.4248.
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.
基于质谱（MS）的高通量筛选（HTS）因其速度、灵敏度、可重复性和无标记读出等特点，作为当前筛选方法的替代方案具有巨大潜力。我们最近报道，新一代基质辅助激光解吸/电离三重四极杆（MALDI-QqQ）质谱仪非常适合各种酶分析和筛选方案。然而，迄今为止测量的所有目标都有肽底物，通过选择离子监测（SIM）可以轻松监测，而不受MALDI基质的干扰。为了进一步将该应用扩展到具有小分子、非肽底物的酶，我们评估了该方法用于测量乙酰胆碱酯酶（AChE）的酶活性和抑制作用。由于存在MALDI基质干扰的可能性，我们研究了多反应监测（MRM）用于选择性MS/MS跃迁，并准确测量乙酰胆碱向胆碱的转化。重要的是，在方法开发过程中通过预平衡MRM跃迁，可以克服底物和产物之间的电离、检测和MRM跃迁效率差异，从而允许使用底物和产物信号的比率直接读出酶活性。该分析方法的进一步验证表明，使用几种已知抑制剂对AChE进行浓度依赖性抑制测量结果准确。最后，以单剂量（10 microM）筛选了一个包含1008种类药物化合物的小型文库，并在二级筛选中对该初步筛选中排名前10的抑制剂进行了验证，以确定每种化合物抑制效力的排名顺序。总体而言，这些数据表明，基于MALDI-QqQMS的读出平台适用于测量小分子底物和产物，并且在速度、灵敏度、可重复性和试剂成本方面比当前的HTS方法具有显著优势。
