Peptide-centric local stability assay (PELSA) for sensitive identification of ligand-targeting proteins and binding...

The peptide-centric local stability assay (PELSA) was developed to identify the protein targets and binding regions of diverse ligands at a proteome-wide scale. In this approach, the ligand is added to a cell lysate, and the proteins are digested for a short, defined time period using 0.5 mg ml^-1 trypsin. Protein regions involved in ligand binding show less digestion, making it possible to identify binding sites and even quantify binding activity by dose-dependent analysis. In contrast to modification-based techniques that require chemical modification of ligands, the ligand modification-free property of the PELSA offers a key advantage by avoiding derivatization, thereby enabling proteome-wide identification of target proteins across diverse ligand types and biological contexts. The PELSA is broadly applicable for identifying protein targets of diverse ligands such as drugs and antibodies as well as endogenous metabolites and metal ions. However, performing PELSA experiments and subsequent data analysis to extract protein-ligand interaction details, including the binding protein, binding region and binding affinity, is not a trivial task. To address this, we present a detailed protocol for the experimental and computational PELSA workflow, including raw data processing and result visualization with the PELSA-Decipher software ( https://zenodo.org/records/18266507 ). Key points, such as selection of ligand concentrations, timing of trypsinization and quality control between different replicates, are discussed. Finally, we demonstrate the application of the protocol for identifying targets of staurosporine and 5-methyltetrahydrofolate, as well as for the dose-dependent PELSA analysis of inhibitors targeting the HSP90 family. The full PELSA protocol can be finished in 2 days, 1 day for sample preparation and 1 day for liquid chromatography-tandem mass spectrometry analysis and data processing. PELSA-Decipher can be downloaded from GitHub ( https://github.com/DICP-1809/ ).