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Probing the Dissociation of Protein Complexes by Means of Gas-Phase H/D Exchange Mass Spectrometry

  • Ulrik H. Mistarz
  • Shane A. Chandler
  • Jeffery M. Brown
  • Justin L. P. BeneschEmail author
  • Kasper D. RandEmail author
Focus: Honoring Carol V. Robinson's Election to the National Academy of Sciences: Research Article

Abstract

Gas-phase hydrogen/deuterium exchange measured by mass spectrometry (gas-phase HDX-MS) is a fast method to probe the conformation of protein ions. The use of gas-phase HDX-MS to investigate the structure and interactions of protein complexes is however mostly unharnessed. Ionizing proteins under conditions that maximize preservation of their native structure (native MS) enables the study of solution-like conformation for milliseconds after electrospray ionization (ESI), which enables the use of ND3-gas inside the mass spectrometer to rapidly deuterate heteroatom-bound non-amide hydrogens. Here, we explored the utility of gas-phase HDX-MS to examine protein-protein complexes and inform on their binding surface and the structural consequences of gas-phase dissociation. Protein complexes ranging from 24 kDa dimers to 395 kDa 24mers were analyzed by gas-phase HDX-MS with subsequent collision-induced dissociation (CID). The number of exchangeable sites involved in complex formation could, therefore, be estimated. For instance, dimers of cytochrome c or α-lactalbumin incorporated less deuterium/subunit than their unbound monomer counterparts, providing a measure of the number of heteroatom-bound side-chain hydrogens involved in complex formation. We furthermore studied if asymmetric charge-partitioning upon dissociation of protein complexes caused intermolecular H/D migration. In larger multimeric protein complexes, the dissociated monomer showed a significant increase in deuterium. This indicates that intermolecular H/D migration occurs as part of the asymmetric partitioning of charge during CID. We discuss several models that may explain this increase deuterium content and find that a model where only deuterium involved in migrating charge can account for most of the deuterium enrichment observed on the ejected monomer. In summary, the deuterium content of the ejected subunit can be used to estimate that of the intact complex with deviations observed for large complexes accounted for by charge migration.

Graphical abstract

Keywords

Hydrogen/deuterium exchange Gas-phase dissociation Protein complexes Structural proteomics 

Notes

Acknowledgements

K.D.R. acknowledges the generous financial support from the Danish Council for Independent Research | Natural Sciences (Steno Grant no. 11-104,058). U.H.M. gratefully acknowledges the COST Action BM1403 for an STSM grant and the Danish Ministry of Higher Education and Science for an Elite Research (EliteForsk) travel grant. S.A.C. and J.L.P.B. thank the Biotechnology and Biological Sciences Research Council (BB/L017067/1) and Waters Corp. for an iCASE studentship.

Supplementary material

13361_2018_2064_MOESM1_ESM.pdf (595 kb)
ESM 1 (PDF 594 kb)

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Copyright information

© American Society for Mass Spectrometry 2018

Authors and Affiliations

  1. 1.Protein Analysis Group, Department of PharmacyUniversity of CopenhagenCopenhagenDenmark
  2. 2.Department of Chemistry, Physical & Theoretical Chemistry LaboratoryUniversity of OxfordOxfordUK
  3. 3.Waters CorporationWilmslowUK

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