Acoustic ejection native mass spectrometry for high-throughput protein-ligand screening

Native mass spectrometry (nMS) enables direct analysis of non-covalent protein-ligand interactions, providing information on binding, stoichiometry, and protein oligomeric state. In drug discovery, nMS is widely used for mechanistic protein-ligand characterization and for confirming hits identified by orthogonal biochemical or biophysical assays. However, limited analytical throughput has restricted its use for screening compound libraries for non-covalent protein-ligand binding. Here, we demonstrate the utility of acoustic ejection mass spectrometry (AEMS) coupled to a high-resolution QTOF mass spectrometer as a high-throughput platform for nMS screening using SARS-CoV-2 main protease (Mpro) as a model protein. Well-resolved charge-state envelopes corresponding to the monomeric and dimeric forms of Mpro enabled simultaneous assessment of ligand binding and changes in oligomeric state. Non-covalent binders, including compounds that perturb the monomer-dimer equilibrium, were readily identified directly from native mass spectra. Analysis of a 1536-well screening plate was completed in approximately 1 h with an acquisition time of 2.5 s per well and excellent reproducibility across the plate. These results demonstrate that acoustic ejection nMS is a practical high-throughput screening platform that simultaneously delivers direct molecular information on protein-ligand binding, binding stoichiometry, and oligomeric distribution.