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.

Identification of Trypanosoma brucei AdoMetDC Inhibitors Using a High-Throughput Mass Spectrometry-Based Assay

“…The assay utilizes the RapidFire instrument (Agilent Technologies), a robotic liquid-handling system with in-line solid-phase extraction (SPE) for rapid mobile phase exchange, interfaced with a triple quadrupole mass spectrometer for quantitative detection of the substrate (AdoMet) and the product (dcAdoMet) of the enzymatic reaction. The RapidFire technology has been used successfully for high-throughput primary screening of enzyme targets otherwise not amenable to rapid testing 30 , including phosphatidylserine decarboxylase 31 .…”

Ultrafast mass spectrometry based bioanalytical method for digoxin supporting an in vitro P‐glycoprotein (P‐gp) inhibition screen

“…The RapidFire™ system made significant improvements over these custombuilt systems by its extensive use of both high-speed robotics and SPE phases with optimized particle and bed sizes. [18] As a result, the RF-MS/MS system is capable of achieving a maximum speed of 5 s per injection for the analysis of in vitro biological samples, which approaches the throughput of a plate-reader but offers a label-free, MS-based, highly selective and sensitive bioanalytical platform for high-throughput applications. An additional advantage that RF-MS/MS enjoys is that it requires either no or very minimal changes in sample preparation as compared to LC-MS/MS analysis, making it easy to be incorporated into existing automated in vitro assays.…”

Mass spectrometry approaches to monitor protein–drug interactions

“…The microfluidics-based multiplexed screen was performed in a high density format (4000 data points on a single MALDI plate) and required only 10 s acquisition time per data point. Similar throughput has recently been reported for an integrated on-line solid-phase extraction system interfaced to a triple-quadrupole mass spectrometer termed RapidFire [42,43]. The selected reaction monitoring capabilities of the triple-quadrupole MS afford improved selectivity, sensitivity and precision of quantification as compared to simple intact mass measurements and ensured compatibility with more complex matrices.…”