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Open AccessArticle

Mass Spectrometric Analysis of Antibody—Epitope Peptide Complex Dissociation: Theoretical Concept and Practical Procedure of Binding Strength Characterization

1
Proteome Center Rostock, University Medicine Rostock, 18059 Rostock, Germany
2
School of Pharmacy, University of Ghana, P. O. Box LG53 Legon, Ghana
*
Author to whom correspondence should be addressed.
Academic Editor: Marilisa Leone
Molecules 2020, 25(20), 4776; https://doi.org/10.3390/molecules25204776
Received: 8 September 2020 / Revised: 9 October 2020 / Accepted: 16 October 2020 / Published: 17 October 2020
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 KD m0g#= 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 KD m0g#= 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 KD m0g#= 4.04 × 10−12 was calculated. Likewise, an apparent KD m0g#= 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. View Full-Text
Keywords: mass spectrometric epitope mapping; gas phase immune complex dissociation; apparent gas phase dissociation constants; apparent gas phase activation energies; ITEM-TWO; native mass spectrometry mass spectrometric epitope mapping; gas phase immune complex dissociation; apparent gas phase dissociation constants; apparent gas phase activation energies; ITEM-TWO; native mass spectrometry
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MDPI and ACS Style

Danquah, B.D.; Opuni, K.F.M.; Roewer, C.; Koy, C.; Glocker, M.O. Mass Spectrometric Analysis of Antibody—Epitope Peptide Complex Dissociation: Theoretical Concept and Practical Procedure of Binding Strength Characterization. Molecules 2020, 25, 4776.

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