High-throughput Mass Spectrometry Based Assays for Detection of Reactive Compounds

• Three mass spectrometry assays detect reactive compounds with distinct mechanisms • Tiered assay approach enables efficient compound triage in screening workflows • High-throughput mass spectrometry reduces solvent waste compared to chromatography High-throughput screening (HTS) is a foundational approach in small molecule drug discovery, but false positives due to reactive compounds can waste resources and hinder progress. Nuisance assays are therefore required to identify such compounds; however, existing methods can suffer from detection artifacts themselves and have limited throughput. Here, we report the development and application of three high-throughput mass spectrometry (HT-MS) based assays: tris(2-carboxyethyl)phosphine (TCEP) oxidation, glutathione (GSH) oxidation, and a La assay to detect reactive molecules (ALARM) by MS. The assays were implemented on an infrared matrix-assisted laser desorption electrospray ionization MS platform. The TCEP and GSH assays, using small molecule surrogates, rapidly detect oxidants, redox cycling compounds, and thiol-reactive compounds, with throughputs of 1–3 minutes per 384-well plate. ALARM MS, a protein-based assay, identifies a broader range of reactive mechanisms, taking approximately 6 minutes per plate. Screening data from over 20,000 compounds demonstrated that TCEP and GSH assays can detect different reactive compound classes. ALARM MS serves as a secondary triage step for hits from high-throughput assays, where a broader range of nuisance mechanisms can be detected. Application of this strategy to an HTS campaign streamlined hit triage and reduced the number of compounds advancing to resource-intensive downstream assays. Our results establish a robust, scalable nuisance assay strategy using HT-MS to efficiently identify and exclude reactive compounds in drug discovery workflows, thereby enhancing screening quality and conserving resources. These assays are readily adaptable to alternative HT-MS platforms and represent valuable tools for modern HTS.