Quantitative assessment of protein structural changes and drug binding affinities using limited proteolysis mass spectrometry

Protein structure is subject to alteration through interactions with various molecules, including protein–protein interactions, aggregation, and binding to other molecular entities. Limited proteolysis coupled with mass spectrometry (LiP-MS) has emerged as a powerful technique for accurately quantifying these structural changes at the peptide level in response to such perturbations. This method enables the simultaneous probing of structural changes across thousands of proteins within a sample. Despite its potential, there is limited validation to confirm whether the quantitative structural changes observed through LiP-MS consistently correspond with these perturbations.

In this study, we investigate the relationship between protein structural changes under drug treatment and the binding affinities of proteins for the drug. More precisely, we demonstrate that the effective dose in dose-response curves derived from LiP-MS data exhibits a linear correlation with drug-protein binding affinity. Moreover, we propose conditions for selecting peptides that show a high linear correlation to binding affinity. The linear relationship is validated through extensive LiP-MS data analysis using various kinase inhibitors with known binding affinities and multiple cancer cell lines.

This work shows the utility of LiP-MS not only as a tool for mapping structural changes in proteins but also as a quantitative approach for assessing drug-protein interactions. By establishing a linear correlation between LiP-MS-derived dose-response data and binding affinities, our findings provide a framework to utilize LiP-MS in drug discovery and development.