Perturbation-Modulated Native Mass Spectrometry Excludes a Nonspecific Drug Target Protein Binder Based on Conformation Stability Change.

Native electrospray ionization mass spectrometry (ESI-MS) is a standard technique for drug-protein screening but may be affected by nonspecific binding. To address this, a reference protein and ligand must be selected for each protein investigated. We report a versatile, informative, high-throughput screening approach to differentiate specific and nonspecific interactions based on charge state distribution (CSD) changes caused by ionization perturbations (e.g., methanol or heat) without the need for a surrogate. We show that specific binding stabilizes the protein-ligand complex against perturbations, resulting in narrower CSDs compared with the unbound protein. In contrast, no significant difference in CSDs is observed between nonspecific complexes and the free protein. To introduce ion source perturbations without affecting protein-ligand incubation, we employed a 3D-printed open port probe (OPP) that is widely compatible with different instruments and sample introduction techniques. The approach was validated with well-characterized protein-ligand pairs, confirming that cytidine phosphates, triacetylchitotriose, and fluvastatin are specific ligands for ribonuclease A, lysozyme, and beta-lactoglobulin, respectively. Further, cytidine-5'-triphosphate (CTP) was found to interact nonspecifically with lysozyme and beta-lactoglobulin. The approach was applied to screening assays of two drug target proteins, thrombin and dihydrofolate reductase, revealing that for thrombin, fluvastatin may share the same binding site as argatroban, which is supported by competition experiments and molecular docking results. These results provide new insights into the anticoagulation effect of statins and show the potential of the approach in prioritizing candidates for target proteins.