Speaker
Description
Systematic mapping of protein-ligand interactions is essential for understanding biological processes and drug mechanisms. By using a limited-proteolysis strategy that employs a large amount of trypsin to generate peptides directly from native proteins, we developed the peptide-centric local stability assay, a modification-free approach that achieves unprecedented sensitivity in proteome-wide target identification and binding-region determination. However, its original workflow is limited in throughput, sample compatibility and accessible protein targets. Recently, we introduce a high-throughput adaptation - HT-PELSA - that increases sample processing efficiency 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, making it possible to identify 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, 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 - offers a powerful platform poised to accelerate both drug discovery and basic biological research.
| Research type | Basic research |
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