Native mass spectrometry enabled by infrared matrix-assisted laser desorption electrospray ionization for rapid measurement of protein-ligand biophysical parameters

The determination of an equilibrium dissociation constant (K_d) alongside the maximum binding capacity (B_max) of protein-ligand interactions is essential in understanding binding affinities and binding capacities which play a major role in the design and development of new therapeutic molecules. Rapid and accurate determination of these biophysical parameters (K_d and B_max) in noncovalent protein-ligand interactions is desirable in the modern drug discovery process. Previously, we demonstrated the detection of noncovalent protein-ligand complexes by infrared-assisted matrix desorption electrospray ionization-mass spectrometry (IR-MALDESI-MS). Here, we report the first determination by IR-MALDESI-MS of biophysical parameters from noncovalent protein-ligand interactions using a ligand titration approach. Unlike conventional electrospray ionization-mass spectrometry (ESI-MS), IR-MALDESI-MS enables analysis in < 13 s per ligand concentration emphasizing its speed, automation, and sensitivity. Native mass spectrometry by IR-MALDESI was performed on carbonic anhydrase II (CAH) incubated with sulfanilamide (SLFA), a known inhibitor. Coupled with other published studies, these results demonstrate that IR-MALDESI has strong potential as a high-throughput screening (HTS) technique for rapidly and accurately determining the K_d and B_max of protein-ligand complexes.