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Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry
Published on: October 15, 2018
Method for stabilizing protein-ligand complexes in nanoelectrospray ionization mass spectrometry
Jiangxiao Sun1, Elena N Kitova, John S Klassen
1Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
Analytical Chemistry
|January 16, 2007
Summary
Researchers successfully detected bovine trypsin and benzamidine complexes using nanoflow electrospray ionization mass spectrometry. Imidazole additive stabilized these ions, enabling accurate binding affinity measurements for protein-ligand interactions.
Area of Science:
- Analytical Chemistry
- Biochemistry
- Physical Chemistry
Background:
- Investigating protein-ligand interactions is crucial for understanding biological processes.
- Previous studies using electrospray ionization mass spectrometry (ES-MS) failed to detect the trypsin-benzamidine complex.
- Gas-phase analysis of non-covalent complexes presents challenges due to ion instability.
Purpose of the Study:
- To investigate the interaction between bovine trypsin and benzamidine in solution and the gas phase.
- To overcome limitations in detecting and stabilizing protein-ligand complexes using mass spectrometry.
- To determine the dissociation pathways and binding affinity of the trypsin-benzamidine complex.
Main Methods:
- Nanoflow electrospray ionization (nanoES) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS).
- Blackbody infrared radiative dissociation (BIRD) technique for time-resolved thermal dissociation experiments.
- Utilizing solution additives to stabilize ions during MS analysis.
Main Results:
- Gaseous protonated ions of the trypsin-benzamidine complex were detected under gentle nanoES conditions.
- Ion lifetimes were short (<0.6 s at >100 °C), indicating dissociation during MS sampling.
- Addition of imidazole to the nanoES solution significantly stabilized the complex ions.
- The association constant (Kassoc) for the trypsin-benzamidine complex was determined to be (1.9 ± 0.2) x 10^4 M⁻¹.
- Imidazole's stabilizing effect was also observed for carbonic anhydrase II and its ligand.
Conclusions:
- Gentle nanoES conditions enable the detection of otherwise unstable protein-ligand complexes.
- Imidazole acts as a stabilizing agent in nanoES-MS, likely through enhanced evaporative cooling.
- This method allows for accurate determination of binding affinities for protein-ligand interactions.
- The findings provide a valuable strategy for analyzing labile non-covalent complexes in the gas phase.
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