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Peptide-centric local stability assay (PELSA) for sensitive identification of ligand targeting proteins and binding sites at proteome scale

ABSTRACT: Recently, a robust ligand modification-free method, termed as peptide-centric local stability assay (PELSA), was developed to identify the protein targets and binding regions of diverse ligands at proteomics scale. This method has unprecedented sensitivity and can be broadly applicable to ligands including drugs, metabolites, metal ions, antibodies etc. However, conduction of PELSA experiment and the extraction of key information on ligand-protein interaction, including the binding protein, binding site, and binding affinity, is not a trivial task. To address this, we introduce a protocol for the conducting of PELSA experiment and the processing of PELSA dataset, including raw data processing and result visualization. Key points, such as selection of ligand concentration, timing of trypsinization, and quality control between different replicates, are also discussed. Finally, we demonstrate how the protocol provided are appropriate for screening the targeting proteins of staurosporine, for screening the targeting proteins of 5-methyltetrahydrofolate, as well as for the dose-dependent PELSA analysis of inhibitors targeting HSP90 family. It takes 2 days to complete the PELSA protocol (additional 8 d to prepare cells), 1 d for sample preparation, 1 d for LC-MS/MS analysis and data processing.

ORGANISM(S): Homo Sapiens

SUBMITTER: Mingliang Ye

PROVIDER: PXD064541 | iProX | Fri May 30 00:00:00 BST 2025

REPOSITORIES: iProX

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