Accelerating multiplexed profiling of protein-ligand interactions: High-throughput plate-based reactive cysteine profiling with minimal input

Chemoproteomics has made significant progress in investigating small-molecule-protein interactions. However, the proteome-wide profiling of cysteine ligandability remains challenging to adapt for high-throughput applications, primarily due to a lack of platforms capable of achieving the desired depth using low input in 96- or 384-well plates. Here, we introduce a revamped, plate-based platform which enables routine interrogation of either similar to 18,000 or similar to 24,000 reactive cysteines based on starting amounts of 10 or 20 mg, respectively. This represents a 5-10X reduction in input and 2-3X improved coverage. We applied the platform to screen 192 electrophiles in the native HEK293T proteome, mapping the ligandability of 38,450 reactive cysteines from 8,274 human proteins. We further applied the platform to characterize new cellular targets of established drugs, uncovering that ARS-1620, a KRAS(G12C) inhibitor, binds to and inhibits an off-target adenosine kinase ADK. The platform represents amajor step forward to high-throughput proteome-wide evaluation of reactive cysteines.