Collision induced unfolding of protein ions in the gas phase studied by ion mobility-mass spectrometry: The effect of ligand binding on conformational stability

  • Jonathan T. S. Hopper
  • Neil J. Oldham


Ion mobility spectrometry, with subsequent mass spectrometric detection, has been employed to study the stability of compact protein conformations of FK-binding protein, hen egg-white lysozyme, and horse heart myoglobin in the presence and absence of bound ligands. Protein ions, generated by electrospray ionization from ammonium acetate buffer, were activated by collision with argon gas to induce unfolding of their compact structures. The collisional cross sections (Ω) of folded and unfolded conformations were measured in the T-Wave mobility cell of a Waters Synapt HDMS (Waters, Altrincham, UK) employing a calibration against literature values for a range of protein standards. In the absence of activation, collisional cross section measurements were found to be consistent with those predicted for folded protein structures. Under conditions of defined collisional activation energies partially unfolded conformations were produced. The degree of unfolding and dissociation induced by these defined collision energies are related to the stability of noncovalent intra- and intermolecular interactions within protein complexes. These findings highlight the additional conformational stability of protein ions in the gas phase resulting from ligand binding.


Lysozyme Collision Energy Collisional Activation Ammonium Acetate Solution Theoretical Cross Section 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Supplementary material

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Supplementary material, approximately 67 KB.


  1. 1.
    Ganem, B.; Li, Y. T.; Henion, J. D. Detection of Noncovalent Receptor Ligand Complexes by Mass-Spectrometry. J. Am. Chem. Soc. 1991, 113, 6294–6296.CrossRefGoogle Scholar
  2. 2.
    Ganem, B.; Li, Y. T.; Henion, J. D. Observation of Noncovalent Enzyme Substrate and Enzyme Product Complexes by Ion-Spray Mass-Spectrometry. J. Am. Chem. Soc. 1991, 113, 7818–7819.CrossRefGoogle Scholar
  3. 3.
    Katta, V.; Chait, B. T. Observation of the Heme Globin Complex in Native Myoglobin by Electrospray-Ionization Mass-Spectrometry. J. Am. Chem. Soc. 1991, 113, 8534–8535.CrossRefGoogle Scholar
  4. 4.
    Loo, J. A. Studying Noncovalent Protein Complexes by Electrospray Ionization Mass Spectrometry. Mass Spectrom. Rev. 1997, 16, 1–23.CrossRefGoogle Scholar
  5. 5.
    Sinz, A. Investigation of Protein-Ligand Interactions by Mass Spectrometry. Chem. Med. Chem. 2007, 2, 425–431.CrossRefGoogle Scholar
  6. 6.
    Kouvatsos, N.; Thurston, V.; Ball, K.; Oldham, N. J.; Thomas, N. R.; Searle, M. S. Bile Acid Interactions with Rabbit Ileal Lipid Binding Protein and an Engineered Helixless Variant Reveal Novel Ligand Binding Properties of a Versatile β-Clam Shell Protein Scaffold. J. Mol. Biol. 2007, 371, 1365–1377.CrossRefGoogle Scholar
  7. 7.
    Oldham, N. J.; Lissina, O.; Nunn, M. A.; Paesen, G. C. Nondenaturing Electrospray Ionization-Mass Spectrometry Reveals Ligand Selectivity in Histamine-Binding Protein RaHBP2. Org. Biomol. Chem. 2003, 1, 3645–3646.CrossRefGoogle Scholar
  8. 8.
    Sobott, F.; Robinson, C. V. Protein Complexes Gain Momentum. Curr. Opin. Struc. Biol. 2002, 12, 729–734.CrossRefGoogle Scholar
  9. 9.
    Veros, C. T.; Oldham, N. J. Quantitative Determination of Lysozyme-Ligand Binding in the Solution and Gas Phases by Electrospray Ionization Mass Spectrometry. Rapid Commun. Mass Spectrom. 2007, 21, 3505–3510.CrossRefGoogle Scholar
  10. 10.
    Mathur, S.; Badertscher, M.; Scott, M.; Zenobi, R. Critical Evaluation of Mass Spectrometric Measurement of Dissociation Constants: Accuracy and Cross-validation against Surface Plasmon Resonance and Circular Dichroism for the Calmodulin-Melittin System. Phys. Chem. Phys. 2007, 9, 6187–6198.CrossRefGoogle Scholar
  11. 11.
    Jecklin, M. C.; Touboul, D.; Bovet, C.; Wortmann, A.; Zenobi, R. Which Electrospray-Based Ionization Method Best Reflects Protein-Ligand Interactions Found In Solution?: A Comparison of ESI, NanoESI, and ESSI for the Determination of Dissociation Constants with Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2008, 19, 1237–1237.CrossRefGoogle Scholar
  12. 12.
    Wortmann, A.; Jecklin, M. C.; Touboul, D.; Badertscher, M.; Zenobi, R. Binding Constant Determination of High-Affinity Protein-Ligand Complexes by Electrospray Ionization Mass Spectrometry and Ligand Competition. J. Mass Spectrom. 2008, 43, 600–608.CrossRefGoogle Scholar
  13. 13.
    Vu, H.; Quinn, R. J. Direct Screening of Natural Product Extracts Using Mass Spectrometry. J. Biomol. Screen. 2008, 13, 265–275.CrossRefGoogle Scholar
  14. 14.
    Poulsen, S. A. Direct Screening of a Dynamic Combinatorial Library Using Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2006, 17, 1074–1080.CrossRefGoogle Scholar
  15. 15.
    Zhou, M.; Sandercock, A. M.; Fraser, C. S.; Ridlova, G.; Stephens, E.; Schenauer, M. R.; Yokoi-Fong, T.; Barsky, D.; Leary, J. A.; Hershey, J. W.; Doudna, J. A.; Robinson, C. V. Mass Spectrometry Reveals Modularity and a Complete Subunit Interaction Map of the Eukaryotic Translation Factor eIF3. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 18139–18144.CrossRefGoogle Scholar
  16. 16.
    Geels, R. B. J.; Calmat, S.; Heck, A. J. R.; Van der Vies, S. M.; Heeren, R. M. A. Thermal Activation of the Cochaperonins GroES and gp31 Probed by Mass Spectrometry. Rapid Commun. Mass Spectrom. 2008, 22, 3633–3641.CrossRefGoogle Scholar
  17. 17.
    Videler, H.; Ilag, L. L.; McKay, A. R. C.; Hanson, C. L.; Robinson, C. V. Mass Spectrometry of Intact Ribosomes. Proceedings of the 130th Nobel Symposium on Molecular Mechanisms in Biological Processes; Tallberg, Sweden, September 2004. Elsevier Science.Google Scholar
  18. 18.
    Rostom, A. A.; Fucini, P.; Benjamin, D. R.; Juenemann, R.; Nierhaus, K. H.; Hartl, F. U.; Dobson, C. M.; Robinson, C. V. Detection and Selective Dissociation of Intact Ribosomes in a Mass Spectrometer. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 5185–5190.CrossRefGoogle Scholar
  19. 19.
    Bothner, B.; Siuzdak, G. Electrospray Ionization of a Whole Virus: Analyzing Mass, Structure, and Viability. Chem. BioChem. 2004, 5, 258–260.Google Scholar
  20. 20.
    Chaplin, M. Opinion—Do We Underestimate the Importance of Water in Cell Biology? Nature Rev. Mol. Cell Biol. 2006, 7, 861–866.CrossRefGoogle Scholar
  21. 21.
    Szep, S.; Park, S.; Boder, E. T.; Van Duyne, G. D.; Saven, J. G. Structural Coupling Between FKBP12 and Buried Water. Prot. Struct. Funct. Bioinformat. 2009, 74, 603–611.CrossRefGoogle Scholar
  22. 22.
    Yin, S.; Xie, Y. M.; Loo, J. A. Mass Spectrometry of Protein-ligand Complexes: Enhanced Gas-Phase Stability of Ribonuclease-Nucleotide complexes. J. Am. Soc. Mass Spectrom. 2008, 19, 1199–1208.CrossRefGoogle Scholar
  23. 23.
    Robinson, C.; Chung, E.; Kragelund, B.; Knudsen, J.; Aplin, R.; Poulsen, F.; Dobson, C. Probing the Nature of Noncovalent Interactions by Mass Spectrometry: A Study of Protein-CoA Ligand Binding and Assembly. J. Am. Chem. Soc. 1996, 118, 8646–8653.CrossRefGoogle Scholar
  24. 24.
    Siuzdak, G.; Bothner, B.; Yeager, M.; Brugidou, C.; Fauquet, C. M.; Hoey, K.; Chang, C. M. Mass Spectrometry and Viral Analysis. Chem. Biol. 1996, 3, 45–48.CrossRefGoogle Scholar
  25. 25.
    Gologan, B.; Takats, Z.; Alvarez, J.; Wiseman, J. M.; Talaty, N.; Ouyang, Z.; Cooks, R. G. Ion Soft-Landing into Liquids: Protein Identification, Separation, and Purification with Retention of Biological Activity. J. Am. Soc. Mass Spectrom. 2004, 15, 1874–1884.CrossRefGoogle Scholar
  26. 26.
    Breuker, K.; McLafferty, F. Stepwise Evolution of Protein Native Structure with Electrospray into the Gas Phase, 10−12 to 102 s. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 18145–18152.CrossRefGoogle Scholar
  27. 27.
    Kanu, A. B.; Dwivedi, P.; Tam, M.; Matz, L.; Hill, H. H. Ion Mobility-Mass Spectrometry. J. Mass Spectrom. 2008, 43, 1–22.CrossRefGoogle Scholar
  28. 28.
    Karasek, F. W. Plasma Chromatography. Anal. Chem. 1974, 46, A710-A720.CrossRefGoogle Scholar
  29. 29.
    Viehland, L. A.; Mason, E. A. Transport Properties of Gaseous-Ions Over a Wide Energy-Range. Atomic Data Nuclear Data Tables. 1995, 60, 37–95.CrossRefGoogle Scholar
  30. 30.
    Creaser, C. S.; Griffiths, J. R.; Bramwell, C. J.; Noreen, S.; Hill, C. A.; Thomas, C. L. P. Ion Mobility Spectrometry: A Review: Part 1. Structural Analysis by Mobility Measurement. Analyst 2004, 129, 984–994.CrossRefGoogle Scholar
  31. 31.
    Clemmer, D. E.; Hudgins, R. R.; Jarrold, M. F. Naked Protein Conformations—Cytochrome c in the Gas Phase. J. Am. Chem. Soc. 1995, 117, 10141–10142.CrossRefGoogle Scholar
  32. 32.
    Valentine, S. J.; Anderson, J. G.; Ellington, A. D.; Clemmer, D. E. Disulfide-Intact and -Reduced Lysozyme in the Gas Phase: Conformations and Pathways of Folding and Unfolding. J. Phys. Chem. B. 1997, 101, 3891–3900.CrossRefGoogle Scholar
  33. 33.
    Badman, E. R.; Myung, S.; Clemmer, D. E. Evidence for Unfolding and Refolding of Gas-Phase Cytochrome c Ions in a Paul Trap. J. Am. Soc. Mass Spectrom. 2005, 16, 1493–1497.CrossRefGoogle Scholar
  34. 34.
    Koeniger, S. L.; Merenbloom, S. I.; Clemmer, D. E. Evidence for Many Resolvable Structures within Conformation Types of Electrosprayed Ubiquitin Ions. J. Phys. Chem. B. 2006, 110, 7017–7021.CrossRefGoogle Scholar
  35. 35.
    Merenbloom, S. I.; Koeniger, S. L.; Bohrer, B. C.; Valentine, S. J.; Clemmer, D. E. Improving the Efficiency of IMS-IMS by a Combing Technique. Anal. Chem. 2008, 80, 1918–1927.CrossRefGoogle Scholar
  36. 36.
    Pringle, S. D.; Giles, K.; Wildgoose, J. L.; Williams, J. P.; Slade, S. E.; Thalassinos, K.; Bateman, R. H.; Bowers, M. T.; Scrivens, J. H. An Investigation of the Mobility Separation of Some Peptide and Protein Ions Using a New Hybrid Quadrupole/Traveling Wave IMS/Oa-TOF Instrument. Int. J. Mass Spectrom. 2007, 261, 1–12.CrossRefGoogle Scholar
  37. 37.
    Ruotolo, B. T.; Giles, K.; Campuzano, I.; Sandercock, A. M.; Bateman, R. H.; Robinson, C. V. Evidence for Macromolecular Protein Rings in the Absence of Bulk Water. Science. 2005, 310, 1658–1661.CrossRefGoogle Scholar
  38. 38.
    Alverdi, V.; Mazon, H.; Versluis, C.; Hemrika, W.; Esposito, G.; van den Heuvel, R.; Scholten, A.; Heck, A. J. R. cGMP-binding Prepares PKG for Substrate Binding by Disclosing the C-Terminal Domain. J. Mol. Biol. 2008, 375, 1380–1393.CrossRefGoogle Scholar
  39. 39.
    Covey, T.; Douglas, D. J. Collision Cross-Sections for Protein Ions. J. Am. Soc. Mass Spectrom. 1993, 4, 616–623.CrossRefGoogle Scholar
  40. 40.
    Thalassinos, K.; Slade, S. E.; Jennings, K. R.; Scrivens, J. H.; Giles, K.; Wildgoose, J.; Hoyes, J.; Bateman, R. H.; Bowers, M. T. Ion Mobility Mass Spectrometry of Proteins in a Modified Commercial Mass Spectrometer. Int. J. Mass Spectrom. 2004, 236, 55–63.CrossRefGoogle Scholar
  41. 41.
    Shvartsburg, A. A.; Jarrold, M. F. An Exact Hard-Spheres Scattering Model for the Mobilities of Polyatomic Ions. Chem. Phys. Lett. 1996, 261, 86–91.CrossRefGoogle Scholar
  42. 42.
    Mesleh, M. F.; Hunter, J. M.; Shvartsburg, A. A.; Schatz, G. C.; Jarrold, M. F. Structural Information from Ion Mobility Measurements: Effects of the Long-Range Potential. J. Phys. Chem. A. 1996, 100, 16082–16086.CrossRefGoogle Scholar
  43. 43.
    Davis, I. W.; Leaver-Fay, A.; Chen, V. B.; Block, J. N.; Kapral, G. J.; Wang, X.; Murray, L. W.; Arendall, W. B.; Snoeyink, J.; Richardson, J. S.; Richardson, D. C. MolProbity: All-Atom Contacts and Structure Validation for Proteins and Nucleic Acids. Nucleic Acids Res. 2007, 35, W375-W383.CrossRefGoogle Scholar
  44. 44.
    Smith, D. P.; Knapman, T. W.; Campuzano, I.; Malham, R. W.; Berryman, J. T.; Radford, S. E.; Ashcroft, A. E. Deciphering Drift Time Measurements from Traveling Wave Ion Mobility Spectrometry-mass Spectrometry Studies. Eur. J. Mass Spectrom. 2009, 15, 113–130.CrossRefGoogle Scholar
  45. 45.
    Mark, K. J.; Douglas, D. J. Coulomb Effects in Binding of Heme in Gas-Phase Ions of Myoglobin. Rapid Commun. Mass Spectrom. 2006, 20, 111–117.CrossRefGoogle Scholar
  46. 46.
    Ghaemmaghami, S.; Fitzgerald, M.; Oas, T. A Quantitative, High-Throughput Screen for Protein Stability. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8296–8301.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2009

Authors and Affiliations

  1. 1.School of ChemistryUniversity of NottinghamNottinghamUK

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