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Abstract
Electrospray mass spectrometry is applied to determine apparent binding energies and quasi equilibrium dissociation constants of immune complex dissociation reactions in the gas phase. Myoglobin, a natural protein-ligand complex, has been used to develop the procedure which starts from determining mean charge states and normalized and averaged ion intensities. The apparent dissociation constant 3.60 × 10−12 for the gas phase heme dissociation process was calculated from the mass spectrometry data and by subsequent extrapolation to room temperature to mimic collision conditions for neutral and resting myoglobin. Similarly, for RNAse S dissociation at room temperature a 4.03 × 10−12 was determined. The protocol was tested with two immune complexes consisting of epitope peptides and monoclonal antibodies. For the epitope peptide dissociation reaction of the FLAG peptide from the antiFLAG antibody complex an apparent gas phase dissociation constant 4.04 × 10−12 was calculated. Likewise, an apparent 4.58 × 10−12 was calculated for the troponin I epitope peptide—antiTroponin I antibody immune complex dissociation. Electrospray mass spectrometry is a rapid method, which requires small sample amounts for either identification of protein-bound ligands or for determination of the apparent gas phase protein-ligand complex binding strengths.
Highlights
IntroductionElectrospray mass spectrometric methods have gained broad acceptance for investigation of the constituents of supramolecular complexes and determination of binding surfaces, e.g., for identifying the locations of partial surfaces on antigens which are recognized by an antibody of interest [1]
= 4.58 × 10−12 was calculated for the troponin I epitope peptide—antiTroponin I antibody m0g immune complex dissociation
Previous reports have shown that high pressure mass spectrometry and/or black body irradiation can be applied for analyzing small molecule-ion equilibria and to determine kinetic and thermodynamic properties, Molecules 2020, 25, 4776; doi:10.3390/molecules25204776
Summary
Electrospray mass spectrometric methods have gained broad acceptance for investigation of the constituents of supramolecular complexes and determination of binding surfaces, e.g., for identifying the locations of partial surfaces on antigens which are recognized by an antibody of interest [1]. Up to now there exists no mass spectrometric method which has gained equal acceptance for investigating gas phase binding strengths of distinct protein-ligand complexes. Gas phase dissociation reactions of Leu-enkephaline dimers [6] and of small proteins, such as ubiquitin [7] had been studied as well. Such investigations included the application of the Eyring–Polanyi equation for bimolecular gas phase reactions [8]. Semi-quantitative analysis of glycan ligand-protein binding has been reported as well to estimate binding strengths by ESI-MS [11]
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