High-throughput peptide-centric local stability assay extends protein–ligand identification to membrane proteins, tissues and bacteria

Abstract Systematic mapping of protein–ligand interactions is essential for understanding biological processes and drug mechanisms. Peptide-centric local stability assay (PELSA) is a powerful tool for detecting these interactions and identifying potential binding sites. However, its original workflow is limited in throughput, sample compatibility and accessible protein targets. Here, we introduce a high-throughput adaptation—HT-PELSA—that increases sample processing efficiency by 100-fold while maintaining high sensitivity and reproducibility. HT-PELSA substantially extends the capabilities of the original method by enabling sensitive protein–ligand profiling in crude cell, tissue and bacterial lysates, allowing the identification of membrane protein targets in diverse biological systems. We demonstrate that HT-PELSA can precisely and accurately determine binding affinities of small molecule inhibitors, sensitively detect direct and allosteric ATP binding sites, and reveal off-target interactions of a marketed kinase inhibitor in heart tissue. By enhancing scalability, reducing costs and enabling system-wide drug screening across a wide range of sample types, HT-PELSA—when combined with next-generation mass spectrometry—may offer a powerful platform poised to accelerate both drug discovery and basic biological research.