Direct Analysis from Phase-Separated Liquid Samples using ADE-OPI-MS: Applicability to High-Throughput Screening for Inhibitors of Diacylglycerol Acyltransferase 2

Abstract

The primary goal of high-throughput screening (HTS) is to rapidly survey a broad collection of compounds, numbering from tens of thousands to millions of members, and identify those that modulate the activity of a therapeutic target of interest. For nearly two decades, mass spectrometry has been used as a label-free, direct-detection method for HTS and is widely acknowledged as being less susceptible to interferences than traditional optical techniques. Despite these advantages, the throughput of conventional MS-based platforms like RapidFire or parallel LC-MS, which typically acquire data at speeds of 6–30 s/sample, can still be limiting for large HTS campaigns. To overcome this bottleneck, the field has recently turned to chromatography-free approaches including MALDI-TOF-MS and acoustic droplet ejection-MS, both of which are capable of throughputs of 1 sample/second or faster. In keeping with these advances, we report here on our own characterization of an acoustic droplet ejection, open port interface (ADE-OPI)-MS system as a platform for HTS using the membrane-associated, lipid metabolizing enzyme diacylglycerol acyltransferase 2 (DGAT2) as a model system. We demonstrate for the first time that the platform is capable of ejecting droplets from phase-separated samples, allowing direct coupling of liquid–liquid extraction with OPI-MS analysis. By applying the platform to screen a 6400-member library, we further demonstrate that the ADE-OPI-MS assay is suitable for HTS and also performs comparably to LC-MS, but with an efficiency gain of >20-fold.

Acoustic ejection mass spectrometry: Development, applications, and future perspective

“…Several groups have demonstrated the use of AEMS in HTS for lead discovery. Wen et al developed an AEMS approach to screen a 6400-member library against lipid-metabolizing enzyme diacylglycerol acyltransferase 2, demonstrating a 20Â gain in efficiency compared to LC-MS (Wen et al, 2021). In addition, they first demonstrated the ability of AEMS to analyze phase-separated samples, which allowed a liquid-liquid extraction to be performed on the biological samples before AEMS readout.…”

“…Finally, to take full advantage of the extremely fast analysis speed of AEMS, it needs to be integrated into fully automated analysis systems equipped with robotic arms, plate hotels, centrifuges, and sealers/de-sealers to enable unattended and continuous operation, as demonstrated by both Simon et al and Wen et al (Simon et al, 2021;Wen et al, 2021). In addition, it is fully expected that AEMS will be integrated into automated assay systems to enable end-to-end assay and analysis operation, which will finally realize our long-pursued dream of an MS-based plate reader for screening applications using AEMS.…”

High-Throughput Mass Spectrometry for Hit Identification: Current Landscape and Future Perspectives

“…More recently, Wen and colleagues reported on their characterization of an ADE-OPI-MS system for the identification of diacylglycerol O -acyltransferase 2 (DGAT2) inhibitors and compared the results to those of the orthogonal fluorescent and LC-MS assay formats. 61 The initial effort utilizing ADE-OPI-MS for the assay suffered from a stability challenge where the triglyceride reaction product nonspecifically adsorbed to the well plate surface. To address this issue, the authors developed an in-well liquid–liquid extraction (LLE) method, adding a pentanol layer on top of the aqueous sample solution.…”

Acoustic Ejection Mass Spectrometry: A Fully Automatable Technology for High-Throughput Screening in Drug Discovery

“…25,26 The concept has recently been investigated by different groups for applicability in the fields of bioanalysis, HTS, parallel organic synthesis, and compound quality control. 27–30 Technical modifications on a prototype system resulted in analytical throughputs of 3 samples/s tracking a single analyte and up to 6 samples/s for different analytes measured from adjacent wells. 31 However, reports to date are limited to small-scale feasibility studies, and a demonstration of HTS capability related to system robustness and automation is still elusive.…”