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

TL;DR

This paper introduces a fast and automated method using IR-MALDESI-MS to measure protein-ligand binding parameters, which could speed up drug discovery.

Contribution

The first use of IR-MALDESI-MS to determine Kd and Bmax for noncovalent protein-ligand interactions via ligand titration.

Findings

IR-MALDESI-MS can analyze protein-ligand interactions in under 13 seconds per concentration.

The method was successfully applied to carbonic anhydrase II and sulfanilamide.

IR-MALDESI shows potential as a high-throughput screening technique for drug discovery.

Abstract

The determination of an equilibrium dissociation constant (Kd) alongside the maximum binding capacity (Bmax) 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 (Kd and Bmax) 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 Kd and Bmax of protein-ligand complexes.

The online version contains supplementary material available at 10.1007/s00216-025-06229-9.

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