Apparent activation energies of protein–protein complex dissociation in the gas–phase determined by electrospray mass spectrometry

Abstract

We have developed a method to determine apparent activation energies of dissociation for ionized protein-protein complexes in the gas phase using electrospray ionization mass spectrometry following the Rice-Ramsperger-Kassel-Marcus quasi-equilibrium theory. Protein-protein complexes were formed in solution, transferred into the gas phase, and separated from excess free protein by ion mobility filtering. Afterwards, complex disassembly was initiated by collision-induced dissociation with step-wise increasing energies. Relative intensities of ion signals were used to calculate apparent activation energies of dissociation in the gas phase by applying linear free energy relations. The method was developed using streptavidin tetramers. Experimentally determined apparent gas-phase activation energies for dissociation ([Formula: see text]) of complexes consisting of Fc parts from immunoglobulins (IgG-Fc) and three closely related protein G' variants (IgG-Fc•protein G'e, IgG-Fc•protein G'f, and IgG-Fc•protein G'g) show the same order of stabilities as can be inferred from their in-solution binding constants. Differences in stabilities between the protein-protein complexes correspond to single amino acid residue exchanges in the IgG-binding regions of the protein G' variants. Graphical abstract Electrospray mass spectrometry and collision-induced dissociation delivers apparent activation energies and supramolecular bond force constants of protein-protein complexes in the gas phase.

Structural Lipids Enable the Formation of Functional Oligomers of the Eukaryotic Purine Symporter UapA

“…Well-resolved spectra showed that the protein exists both as a monomer and a dimer ( Figures 1 A and 1B). The relative abundance of monomer/dimer was ∼5:95, suggesting a strong dimer interface ( Yefremova et al., 2017 ). The spectra also revealed the binding of two adduct molecules (measured mass: 775 ± 50 Da) to the UapAG411V Δ1-11 dimer.…”

Trajectory Taken by Dimeric Cu/Zn Superoxide Dismutase through the Protein Unfolding and Dissociation Landscape Is Modulated by Salt Bridge Formation

“…We have demonstrated how insight into the structural changes that SOD1 undergoes upon gas phase activation, highlighting how both bond breakage and formation pay a role in governing the trajectory taken and the products observed. We have presented an Arrhenius-type framework for analysis which, despite assumptions implicit therein and together with other recently presented methodologies 58 , represents a means for meaningful and direct comparison between proteins in terms of the barriers on the free energy landscape. This work therefore represents a step towards maximising the utility of information extracted from native MS experiments where the protein folding funnel is explored by deliberate gas phase activation 23 .…”

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

“…Interestingly, from ESI-MS ETD studies of multiply charged myoglobin gas phase ions it was concluded that—depending on the complexes´ charge states—the heme group might be coordinated by one of two histidinyl residues, mainly by His93 but also by His64, or by both, suggesting some similarity between in-solution and gas phase complex structures—at least around the heme binding pocket [ 25 ]. The existence of relatively defined macromolecular structures during the heme dissociation process (e.g., as transition state) fits our model, so we postulate that dissociation of immune complexes follows in principle a hard spheres model, i.e., entropy contributions at the transition state are small [ 14 ]. In fact, in-solution antibody—antigen interactions are enthalpy-driven [ 28 , 29 ].…”

“…The uncertainty of this method has been estimated to be approx. 10% [ 14 ]. Hence, myoglobin is considered to be an adequate standard for developing the ESI-MS method by which protein-ligand dissociation reactions may be studied.…”

“…Recently, and still uncommonly, the combination of fast and robust gas-phase epitope mapping methods [ 12 , 13 ] with mass spectrometry-based determination of quasi-thermodynamic information has been published. The latter was obtained based on desolvated and multiply charged and accelerated protein-protein complex ions in the gas phase [ 14 ]. These studies, together with reports on collision induced unfolding reactions of protein ions [ 15 ], have enabled the development of a method termed ITEM-TWO (Intact Transition Epitope Mapping—Thermodynamic Weak-force Order) [ 16 ] that can simultaneously identify epitopes as well as enables to determine gas phase binding strengths of the respective antibody-epitope peptide interactions.…”