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

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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 (\( {E}_{A\ m0g}^{\#} \)) 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.

Electrospray mass spectrometry and collision-induced dissociation delivers apparent activation energies and supramolecular bond force constants of protein-protein complexes in the gas phase.

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Abbreviations

ESI:

electrospray ionization

\( {E}_{A\ m0g}^{\#} \) :

EA: energy of activation, #: apparent (with merged temperature term), m: mean of charge states, 0: at Ecom = 0 eV, g: gas phase

Fc part:

fragment crystallizable part

IgG:

immunoglobulin G

IVIG:

intravenous immunoglobulin

KD s :

dissociation constant in solution

\( {K}_{D\ m0g}^{\#} \) :

KD: dissociation constant, #: apparent (with merged temperature term), m: mean of charge states, 0: at Ecom = 0 eV, g: gas phase

LFE:

linear free energy

Protein G′:

protein G prime

Protein G′e:

protein G prime e (extended)

Protein G′f:

protein G prime f

Protein G′g:

protein G prime g

ToF:

time of flight

ΔG s :

Gibbs free energy difference in solution

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Acknowledgements

We express our thanks to Dr. Stephan Mikkat for providing expertise on mass spectrometry and to Ms Ursula Glocker for preliminary technical assistance. We also thank Dr. Marcus Frank for providing access to the capillary sputter. We acknowledge the German Academic Exchange Service (DAAD) for providing scholarships for YY (no. 91523785), BD (no. 91566064), and KO (no. 91548123). The WATERS Synapt G2S mass spectrometer has been bought through an EU grant (EFRE-UHROM 9) made available to MOG.

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Author notes

  1. Kwabena F.M. Opuni

    Present address: School of Pharmacy, University of Ghana, P.O. Box LG43, Legon Accra, Ghana

  2. Alexandra Ehrens

    Present address: University Hospital of Bonn, Sigmung-Freud-Str. 25, 53105, Bonn, Germany

  3. Yelena Yefremova and F. Teresa I. Melder contributed equally to this work.

Authors and Affiliations

  1. Proteome Center Rostock, University Rostock Medical Center, Schillingallee 69, 18059, Rostock, Germany

    Yelena Yefremova, F. Teresa I. Melder, Bright D. Danquah, Kwabena F.M. Opuni, Cornelia Koy & Michael O. Glocker

  2. University of Applied Sciences Bonn-Rhein-Sieg, von-Liebig-Str. 20, 53359, Rheinbach, Germany

    Alexandra Ehrens, David Frommholz & Harald Illges

  3. Department of Chemistry, University of Antwerp, Groenenborgerlaan 171, 2020, Antwerp, Belgium

    Knut Koelbel & Frank Sobott

  4. Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK

    Frank Sobott

  5. School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK

    Frank Sobott

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  1. Yelena Yefremova
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Correspondence to Michael O. Glocker.

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Yefremova, Y., Melder, F.T.I., Danquah, B.D. et al. Apparent activation energies of protein–protein complex dissociation in the gas–phase determined by electrospray mass spectrometry. Anal Bioanal Chem 409, 6549–6558 (2017). https://doi.org/10.1007/s00216-017-0603-4

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