High Throughput Screening via Mass Spectrometry: A Case Study Using Acetylcholinesterase

Abstract

Mass spectrometry-based screening can be applied to a wide range of targets, including those intractable targets that use substrates such as lipids, fatty acids, phospholipids, steroids, prostaglandins, and other compounds not generally amenable to conventional screening techniques. The major limitation to this approach is throughput, making HTS via mass spectrometry impractical. We present a mass spectrometry-based technique and hardware for lead discovery applications. Mass spectrometry enables the design of label-free assays using biologically native substrates for a wide range of enzymatic targets. This system can be used for the direct quantification of analytes in complex reaction mixtures with typical throughputs of 4-5 s per sample. A mass spectrometry-based assay was developed to identify inhibitors of acetylcholinesterase, an enzyme with clinical importance in Alzheimer's disease. The system was used to screen a small chemical library. Several potent inhibitors were identified, and the IC(50) values of the inhibitors were determined.

Rapid and label-free screening of enzyme inhibitors using segmented flow electrospray ionization mass spectrometry

“…The analysis rate of the segmented flow method compares favorably with previously reported flow injection AchE assays [26,27,29]. The speed of flow injection methods was limited by the need to inject individual samples or additional separation steps when assay buffer was not directly compatible with ESI-MS.…”

“…It has been demonstrated that AchE assays can be performed using flow-injection ESI-MS [26] and HPLC-MS [27] to directly detect substrate and/or product of the reaction. Throughput of 0.2 Hz with 1 to 5 L of sample consumption was possible when using an automated sampling and injection system [27]. In this work, we demonstrate that with direct ESI-MS analysis of segmented assay mixtures, we can generate a throughput of 0.65 Hz for AchE inhibitor screening while consuming 10 nL of sample.…”

Monitoring enzyme reaction and screening of inhibitors of acetylcholinesterase by quantitative matrix-assisted laser desorption/ionization fourier transform mass spectrometry

“…In recent years, mass spectrometry (MS)-based assays have attracted great attention for high-throughput screening. MS offers the significant advantage that it does not require analytes to be labeled, either by direct attachment of fluorescent and radioactive labels or by binding of antibodies, and therefore offers greater flexibility in experiments [12][13][14]. In the past decade, considerable effort was invested to develop MS-based detection schemes capable of assaying inhibition of enzyme-mediated reactions [15,16].…”

Demonstrating Enhanced Throughput of RapidFire Mass Spectrometry through Multiplexing Using the JmjD2d Demethylase as a Model System

“…It offers a direct, label-free readout of product formation with reduced risk of interference compared with alternatives and no requirement for antibody generation. 16,17 However, the capacity of this system renders it a medium-throughput rather than a high-throughput technology. Fluorescent-based methods are therefore still employed for diversity screening of these targets.…”

“…We have previously reported on the application of RapidFire (Agilent Technologies, Lexington, MA) high-throughput mass spectrometry to screening of ~100,000 compounds against JmjD2c. 15 However, despite the throughput advances of the RapidFire system relative to liquid chromatography-mass spectrometry (sample times of ~8 s/well), 16,17 the system is still not adequate for largescale HTS in a reasonable time frame. One could enable HTS by RapidFire with access to multiple systems in parallel; however, it is unlikely that capital investment in a single technology at this scale would occur within the pharmaceutical industry today.…”