Collision-Induced Unfolding Is Sensitive to the Polarity of Proteins and Protein Complexes

  • Seoyeon Hong
  • Matthew F. BushEmail author
Research Article


Collision-induced unfolding (CIU) uses ion mobility to probe the structures of ions of proteins and noncovalent complexes as a function of the extent of gas-phase activation prior to analysis. CIU can be sensitive to domain structures, isoform identities, and binding partners, which makes it appealing for many applications. Almost all previous applications of CIU have probed cations. Here, we evaluate the application of CIU to anions and compare the results for anions with those for cations. Towards that end, we developed a “similarity score” that we used to quantify the differences between the results of different CIU experiments and evaluate the significance of those differences relative to the variance of the underlying measurements. Many of the differences between anions and cations that were identified can be attributed to the lower absolute charge states of anions. For example, the extents of the increase in collision cross section over the full range of energies depended strongly on absolute charge state. However, over intermediate energies, there are significant difference between anions and cations with the same absolute charge state. Therefore, CIU is sensitive to the polarity of protein ions. Based on these results, we propose that the utility of CIU to differentiate similar proteins or noncovalent complexes may also depend on polarity. More generally, these results indicate that the relationship between the structures and dynamics of native-like cations and anions deserve further attention and that future studies may benefit from integrating results from ions of both polarities.


Ion mobility Collision-induced unfolding Protein structure 



This material is based upon work supported by the National Science Foundation under CHE-1807382 (M. F. B.).

Supplementary material

13361_2019_2326_MOESM1_ESM.pdf (1.6 mb)
ESM 1 (PDF 1587 kb)


  1. 1.
    Stengel, F., Baldwin, A.J., Bush, M.F., Hilton, G.R., Lioe, H., Basha, E., Jaya, N., Vierling, E., Benesch, J.L.P.: Dissecting heterogeneous molecular chaperone complexes using a mass spectrum deconvolution approach. Chem. Biol. 19, 599–607 (2012)CrossRefGoogle Scholar
  2. 2.
    Heck, A.J.R.: Native mass spectrometry: a bridge between interactomics and structural biology. Nat. Methods. 5, 927–933 (2008)CrossRefGoogle Scholar
  3. 3.
    El-Hawiet, A., Kitova, E.N., Arutyunov, D., Simpson, D.J., Szymanski, C.M., Klassen, J.S.: Quantifying ligand binding to large protein complexes using electrospray ionization mass spectrometry. Anal. Chem. 84, 3867–3870 (2012)CrossRefGoogle Scholar
  4. 4.
    Uetrecht, C., Versluis, C., Watts, N.R., Roos, W.H., Wuite, G.J.L., Wingfield, P.T., Steven, A.C., Heck, A.J.R.: High-resolution mass spectrometry of viral assemblies: molecular composition and stability of dimorphic hepatitis B virus capsids. Proc. Natl. Acad. Sci. 105, 9216–9220 (2008)CrossRefGoogle Scholar
  5. 5.
    Bereszczak, J.Z., Havlik, M., Weiss, V.U., Marchetti-Deschmann, M., van Duijn, E., Watts, N.R., Wingfield, P.T., Allmaier, G., Steven, A.C., Heck, A.J.R.: Sizing up large protein complexes by electrospray ionisation-based electrophoretic mobility and native mass spectrometry: morphology selective binding of Fabs to hepatitis B virus capsids. Anal. Bioanal. Chem. 406, 1437–1446 (2014)CrossRefGoogle Scholar
  6. 6.
    Uetrecht, C., Barbu, I.M., Shoemaker, G.K., van Duijn, E., Heck, A.J.R.: Interrogating viral capsid assembly with ion mobility–mass spectrometry. Nat. Chem. 3, 126 (2010)CrossRefGoogle Scholar
  7. 7.
    Lutomski, C.A., Lyktey, N.A., Zhao, Z., Pierson, E.E., Zlotnick, A., Jarrold, M.F.: Hepatitis B virus capsid completion occurs through error correction. J. Am. Chem. Soc. 139, 16932–16938 (2017)CrossRefGoogle Scholar
  8. 8.
    Konermann, L., Douglas, D.J.: Unfolding of proteins monitored by electrospray ionization mass spectrometry: a comparison of positive and negative ion modes. J. Am. Soc. Mass Spectrom. 9, 1248–1254 (1998)CrossRefGoogle Scholar
  9. 9.
    Heck, A.J.R., van den Heuvel, R.H.H.: Investigation of intact protein complexes by mass spectrometry. Mass Spectrom. Rev. 23, 368–389 (2004)CrossRefGoogle Scholar
  10. 10.
    Allen, S.J., Schwartz, A.M., Bush, M.F.: Effects of polarity on the structures and charge states of native-like proteins and protein complexes in the gas phase. Anal. Chem. 85, 12055–12061 (2013)CrossRefGoogle Scholar
  11. 11.
    Liko, I., Hopper, J.T.S., Allison, T.M., Benesch, J.L.P., Robinson, C.V.: Negative ions enhance survival of membrane protein complexes. J. Am. Soc. Mass Spectrom. 27, 1099–1104 (2016)CrossRefGoogle Scholar
  12. 12.
    Hogan, C.J., Carroll, J.A., Rohrs, H.W., Biswas, P., Gross, M.L.: Combined charged residue-field emission model of macromolecular electrospray ionization. Anal. Chem. 81, 369–377 (2009)CrossRefGoogle Scholar
  13. 13.
    Loo, R.R.O., Loo, J.A.: Salt bridge rearrangement (SaBRe) explains the dissociation behavior of noncovalent complexes. J. Am. Soc. Mass Spectrom. 27, 975–990 (2016)CrossRefGoogle Scholar
  14. 14.
    Konermann, L.: Molecular dynamics simulations on gas-phase proteins with mobile protons: inclusion of all-atom charge solvation. J. Phys. Chem. B. 121, 8102–8112 (2017)CrossRefGoogle Scholar
  15. 15.
    Laszlo, K.J., Bush, M.F.: Interpreting the collision cross sections of native-like protein ions: insights from cation-to-anion proton-transfer reactions. Anal. Chem. 89, 7607–7614 (2017)CrossRefGoogle Scholar
  16. 16.
    Benesch, J.L.P.: Collisional activation of protein complexes: picking up the pieces. J. Am. Soc. Mass Spectrom. 20, 341–348 (2009)CrossRefGoogle Scholar
  17. 17.
    Dixit, S.M., Polasky, D.A., Ruotolo, B.T.: Collision induced unfolding of isolated proteins in the gas phase: past, present, and future. Curr. Opin. Chem. Biol. 42, 93–100 (2018)CrossRefGoogle Scholar
  18. 18.
    Shelimov, K.B., Jarrold, M.F.: Conformations, unfolding, and refolding of apomyoglobin in vacuum: an activation barrier for gas-phase protein folding. J. Am. Chem. Soc. 119, 2987–2994 (1997)CrossRefGoogle Scholar
  19. 19.
    Pagel, K., Hyung, S.-J., Ruotolo, B.T., Robinson, C.V.: Alternate dissociation pathways identified in charge-reduced protein complex ions. Anal. Chem. 82, 5363–5372 (2010)CrossRefGoogle Scholar
  20. 20.
    Hall, Z., Politis, A., Bush, M.F., Smith, L.J., Robinson, C.V.: Charge-state dependent compaction and dissociation of protein complexes: insights from ion mobility and molecular dynamics. J. Am. Chem. Soc. 134, 3429–3438 (2012)CrossRefGoogle Scholar
  21. 21.
    Zhong, Y., Han, L., Ruotolo, B.T.: Collisional and coulombic unfolding of gas-phase proteins: high correlation to their domain structures in solution. Angew. Chem. Int. Ed. 53, 9209–9212 (2014)CrossRefGoogle Scholar
  22. 22.
    Tian, Y., Han, L., Buckner, A.C., Ruotolo, B.T.: Collision induced unfolding of intact antibodies: rapid characterization of disulfide bonding patterns, glycosylation, and structures. Anal. Chem. 87, 11509–11515 (2015)CrossRefGoogle Scholar
  23. 23.
    Hopper, J.T.S., Oldham, N.J.: Collision induced unfolding of protein ions in the gas phase studied by ion mobility-mass spectrometry: the effect of ligand binding on conformational stability. J. Am. Soc. Mass Spectrom. 20, 1851–1858 (2009)CrossRefGoogle Scholar
  24. 24.
    Hyung, S.-J., Robinson, C.V., Ruotolo, B.T.: Gas-phase unfolding and disassembly reveals stability differences in ligand-bound multiprotein complexes. Chem. Biol. 16, 382–390 (2009)CrossRefGoogle Scholar
  25. 25.
    Rabuck, J.N., Hyung, S.-J., Ko, K.S., Fox, C.C., Soellner, M.B., Ruotolo, B.T.: Activation state-selective kinase inhibitor assay based on ion mobility-mass spectrometry. Anal. Chem. 85, 6995–7002 (2013)CrossRefGoogle Scholar
  26. 26.
    Zhao, Y., Yang, J.Y., Thieker, D.F., Xu, Y., Zong, C., Boons, G.-J., Liu, J., Woods, R.J., Moremen, K.W., Amster, I.J.: A traveling wave ion mobility spectrometry (TWIMS) study of the Robo1-heparan sulfate interaction. J. Am. Soc. Mass Spectrom. 29, 1153–1165 (2018)CrossRefGoogle Scholar
  27. 27.
    Rabuck-Gibbons, J.N., Lodge, J.M., Mapp, A.K., Ruotolo, B.T.: Collision-induced unfolding reveals unique fingerprints for remote protein interaction sites in the KIX regulation domain. J. Am. Soc. Mass Spectrom. (2018)Google Scholar
  28. 28.
    Wagner, N.D., Clemmer, D.E., Russell, D.H.: ESI-IM-MS and collision-induced unfolding that provide insight into the linkage-dependent interfacial interactions of covalently linked diubiquitin. Anal. Chem. 89, 10094–10103 (2017)CrossRefGoogle Scholar
  29. 29.
    Zhang, Y., Deng, L., Kitova, E.N., Klassen, J.S.: Dissociation of multisubunit protein–ligand complexes in the gas phase. Evidence for ligand migration. J. Am. Soc. Mass Spectrom. 24, 1573–1583 (2013). doi:Google Scholar
  30. 30.
    Davidson, K.L., Oberreit, D.R., Hogan, C.J., Bush, M.F.: Nonspecific aggregation in native electrokinetic nanoelectrospray ionization. Int. J. Mass Spectrom. 420, 34–42 (2016)Google Scholar
  31. 31.
    Giles, K., Williams, J.P., Campuzano, I.: Enhancements in travelling wave ion mobility resolution. Rapid Commun. Mass Spectrom. 25, 1559–1566 (2011)CrossRefGoogle Scholar
  32. 32.
    Allen, S.J., Giles, K., Gilbert, T., Bush, M.F.: Ion mobility mass spectrometry of peptide, protein, and protein complex ions using a radio-frequency confining drift cell. Analyst. 141, 884–891 (2016)CrossRefGoogle Scholar
  33. 33.
    Bush, M.F., Campuzano, I.D.G., Robinson, C.V.: Ion mobility mass spectrometry of peptide ions: effects of drift gas and calibration strategies. Anal. Chem. 84, 7124–7130 (2012)CrossRefGoogle Scholar
  34. 34.
    Freeke, J., Bush, M.F., Robinson, C.V., Ruotolo, B.T.: Gas-phase protein assemblies: unfolding landscapes and preserving native-like structures using noncovalent adducts. Chem. Phys. Lett. 524, 1–9 (2012)CrossRefGoogle Scholar
  35. 35.
    McClory, P.J., Håkansson, K.: Corona discharge suppression in negative ion mode nanoelectrospray ionization via trifluoroethanol addition. Anal. Chem. 89, 10188–10193 (2017)CrossRefGoogle Scholar
  36. 36.
    Laskin, J., Futrell, J.H.: Activation of large ions in FT-ICR mass spectrometry. Mass Spectrom. Rev. 24, 135–167 (2005)CrossRefGoogle Scholar
  37. 37.
    Zhuang, X., Gavriilidou, A.F.M., Zenobi, R.: Influence of alkylammonium acetate buffers on protein–ligand noncovalent interactions using native mass spectrometry. J. Am. Soc. Mass Spectrom. 28, 341–346 (2017)CrossRefGoogle Scholar
  38. 38.
    Polasky, D.A., Dixit, S.M., Fantin, S.M., Ruotolo, B.T.: CIUSuite 2: next-generation software for the analysis of gas-phase protein unfolding data. Anal. Chem. 91, 3147–3155 (2019)CrossRefGoogle Scholar
  39. 39.
    Han, L., Hyung, S.-J., Mayers, J.J.S., Ruotolo, B.T.: Bound anions differentially stabilize multiprotein complexes in the absence of bulk solvent. J. Am. Chem. Soc. 133, 11358–11367 (2011)CrossRefGoogle Scholar
  40. 40.
    Han, L., Hyung, S.-J., Ruotolo, B.T.: Bound cations significantly stabilize the structure of multiprotein complexes in the gas-phase. Angew. Chem. Int. Ed Engl. 51, 5692–5695 (2012)CrossRefGoogle Scholar
  41. 41.
    DeLange, R.J., Huang, T.S.: Egg white avidin. 3. Sequence of the 78-residue middle cyanogen bromide peptide. Complete amino acid sequence of the protein subunit. J. Biol. Chem. 246, 698–709 (1971)Google Scholar
  42. 42.
    Lössl, P., Snijder, J., Heck, A.J.R.: Boundaries of mass resolution in native mass spectrometry. J. Am. Soc. Mass Spectrom. 25, 906–917 (2014)CrossRefGoogle Scholar
  43. 43.
    Benesch, J.L.P., Ruotolo, B.T., Simmons, D.A., Robinson, C.V.: Protein complexes in the gas phase: technology for structural genomics and proteomics. Chem. Rev. 107, 3544–3567 (2007)CrossRefGoogle Scholar
  44. 44.
    Sobott, F., McCammon, M.G., Robinson, C.V.: Gas-phase dissociation pathways of a tetrameric protein complex. Int. J. Mass Spectrom. 230, 193–200 (2003)CrossRefGoogle Scholar
  45. 45.
    Felitsyn, N., Kitova, E.N., Klassen, J.S.: Thermal decomposition of a gaseous multiprotein complex studied by blackbody infrared radiative dissociation. Investigating the origin of the asymmetric dissociation behavior. Anal. Chem. 73, 4647–4661 (2001)CrossRefGoogle Scholar
  46. 46.
    Jurchen, J.C., Williams, E.R.: Origin of asymmetric charge partitioning in the dissociation of gas-phase protein homodimers. J. Am. Chem. Soc. 125, 2817–2826 (2003)CrossRefGoogle Scholar
  47. 47.
    Jurchen, J.C., Garcia, D.E., Williams, E.R.: Further studies on the origins of asymmetric charge partitioning in protein homodimers. J. Am. Soc. Mass Spectrom. 15, 1408–1415 (2004)CrossRefGoogle Scholar
  48. 48.
    Fegan, S.K., Thachuk, M.: A charge moving algorithm for molecular dynamics simulations of gas-phase proteins. J. Chem. Theory Comput. 9, 2531–2539 (2013)CrossRefGoogle Scholar
  49. 49.
    Popa, V., Trecroce, D.A., McAllister, R.G., Konermann, L.: Collision-induced dissociation of electrosprayed protein complexes: an all-atom molecular dynamics model with mobile protons. J. Phys. Chem. B. 120, 5114–5124 (2016)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2019

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of WashingtonSeattleUSA

Personalised recommendations