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<p>The determination of an equilibrium dissociation constant (<i>K</i><sub>d</sub>) alongside the maximum binding capacity (<i>B</i><sub>max</sub>) 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 (<i>K</i><sub>d</sub> and <i>B</i><sub>max</sub>) 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 &lt; 13&#xa0;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 <i>K</i><sub>d</sub> and <i>B</i><sub>max</sub> of protein-ligand complexes.</p> Graphical abstract <p></p>

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

  • 期刊论文

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  • 发表日期：

    2025年11月14日

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  • Adeleke A. Adepoju,
  • Reza A. Ghiladi,
  • David C. Muddiman

摘要

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.

Graphical abstract