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

We describe theoretical concepts and explain practical procedures of an electrospray mass spectrometry method, termed “Intact Transmission Epitope Mapping – Thermodynamic Weak-force Order (ITEM-TWO)”, by which can be determined apparent binding energies and dissociation constants of immune complex dissociation reactions in the gas phase. The protocol was developed using natural protein-ligand complexes (myoglobin and RNAse S) and was tested with two immune complexes (FLAG peptide - antiFLAG antibody and Troponin I epitope peptide - anti-Troponin I antibody). ITEM-TWO is a rapid method which requires very little sample consumption for identification of protein bound ligands and for determination of the gas phase protein-ligand complex binding strength.