Characterization of Amyloid Oligomers by Electrospray Ionization-Ion Mobility Spectrometry-Mass Spectrometry (ESI-IMS-MS)

  • Charlotte A. Scarff
  • Alison E. Ashcroft
  • Sheena E. Radford
Part of the Methods in Molecular Biology book series (MIMB, volume 1345)


Soluble oligomers formed during the self-assembly of amyloidogenic peptide and protein species are generally thought to be highly toxic. Consequently, thorough characterization of these species is of much interest in the quest for effective therapeutics and for an enhanced understanding of amyloid fibrillation pathways. The structural characterization of oligomeric species, however, is challenging as they are often transiently and lowly populated, and highly heterogeneous. Electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) is a powerful technique which is able to detect individual ion species populated within a complex heterogeneous mixture and characterize them in terms of shape, stoichiometry, ligand binding capability, and relative stability. Herein, we describe the use of ESI-IMS-MS to characterize the size and shape of oligomers of beta-2-microglobulin through use of data calibration and the derivation of models. This enables information about the range of oligomeric species populated en route to amyloid formation and the mode of oligomer growth to be obtained.

Key words

Protein aggregation Amyloid Oligomerization Native mass spectrometry Ion mobility spectrometry-mass spectrometry 



CAS is funded by the Biotechnology and Biological Sciences Research Council (BBSRC; grant number BB/H024875/1). The Synapt HDMS mass spectrometer was purchased with funds from the BBSRC’s Research Equipment Initiative (BB/E012558/1). SER’s research is supported by an ERC Advanced grant (322408). The authors would like to thank Dr. Anton Calabrese for critical evaluation of the manuscript.


  1. 1.
    Woods LA, Radford SE, Ashcroft AE (2013) Advances in ion mobility spectrometry–mass spectrometry reveal key insights into amyloid assembly. Biochim Biophys Acta 1834:1257–1268PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Hilton GR, Benesch JLP (2012) Two decades of studying non-covalent biomolecular assemblies by means of electrospray ionization mass spectrometry. J R Soc Interface 9:801–816PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Ruotolo BT, Robinson CV (2006) Aspects of native proteins are retained in vacuum. Curr Opin Chem Biol 10:402–408CrossRefPubMedGoogle Scholar
  4. 4.
    Ruotolo BT, Giles K, Campuzano I et al (2005) Evidence for macromolecular protein rings in the absence of bulk water. Science 310:1658–1661CrossRefPubMedGoogle Scholar
  5. 5.
    Smith DP, Radford SE, Ashcroft AE (2010) Elongated oligomers in β2-microglobulin amyloid assembly revealed by ion mobility spectrometry-mass spectrometry. Proc Natl Acad Sci U S A 107:6794–6798PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Smith DP, Giles K, Bateman RH et al (2007) Monitoring copopulated conformational states during protein folding events using electrospray ionization-ion mobility spectrometry-mass spectrometry. J Am Soc Mass Spectrom 18:2180–2190PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Smith DP, Woods LA, Radford SE et al (2011) Structure and dynamics of oligomeric intermediates in β2-microglobulin self-assembly. Biophys J 101:1238–1247PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Woods LA, Platt GW, Hellewell AL et al (2011) Ligand binding to distinct states diverts aggregation of an amyloid-forming protein. Nat Chem Biol 7:730–739PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Young LM, Cao P, Raleigh DP et al (2013) Ion mobility spectrometry–mass spectrometry defines the oligomeric intermediates in amylin amyloid formation and the mode of action of inhibitors. J Am Chem Soc 136:660–670PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Pringle SD, Giles K, Wildgoose JL et al (2007) An investigation of the mobility separation of some peptide and protein ions using a new hybrid quadrupole/travelling wave ims/oa-tof instrument. Int J Mass Spectrom 261:1–12CrossRefGoogle Scholar
  11. 11.
    Ruotolo BT, Benesch JLP, Sandercock AM et al (2008) Ion mobility-mass spectrometry analysis of large protein complexes. Nat Protoc 3:1139–1152CrossRefPubMedGoogle Scholar
  12. 12.
    Scarff CA, Thalassinos K, Hilton GR et al (2008) Travelling wave ion mobility mass spectrometry studies of protein structure: biological significance and comparison with x-ray crystallography and nuclear magnetic resonance spectroscopy measurements. Rapid Commun Mass Spectrom 22:3297–3304CrossRefPubMedGoogle Scholar
  13. 13.
    Smith DP, Knapman TW, Campuzano I et al (2009) Deciphering drift time measurements from travelling wave ion mobility spectrometry-mass spectrometry studies. Eur J Mass Spectrom 15:113–130CrossRefGoogle Scholar
  14. 14.
    Bleiholder C, Dupuis NF, Wyttenbach T et al (2011) Ion mobility–mass spectrometry reveals a conformational conversion from random assembly to β-sheet in amyloid fibril formation. Nat Chem 3:172–177PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Lanucara F, Holman SW, Gray CJ et al (2014) The power of ion mobility-mass spectrometry for structural characterization and the study of conformational dynamics. Nat Chem 6:281–294CrossRefPubMedGoogle Scholar
  16. 16.
    Zhong Y, Han L, Ruotolo BT (2014) Collisional and coulombic unfolding of gas-phase proteins: high correlation to their domain structures in solution. Angew Chem Int Ed Engl 53:9209. doi: 10.1002/anie.201403784 CrossRefPubMedGoogle Scholar
  17. 17.
    Leney AC, Pashley CL, Scarff CA et al (2014) Insights into the role of the beta-2 microglobulin d-strand in amyloid propensity revealed by mass spectrometry. Mol Biosyst 10:412–420PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    Eichner T, Kalverda AP, Thompson GS et al (2011) Conformational conversion during amyloid formation at atomic resolution. Mol Cell 41:161–172PubMedCentralCrossRefPubMedGoogle Scholar
  19. 19.
    Sarell CJ, Woods LA, Su Y et al (2013) Expanding the repertoire of amyloid polymorphs by co-polymerization of related protein precursors. J Biol Chem 288:7327–7337PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Bellotti V, Stoppini M, Mangione P et al (1998) β2-microglobulin can be refolded into a native state from ex vivo amyloid fibrils. Eur J Biochem 258:61–67CrossRefPubMedGoogle Scholar
  21. 21.
    Hernandez H, Robinson CV (2007) Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry. Nat Protoc 2:715–726CrossRefPubMedGoogle Scholar
  22. 22.
    Chernushevich IV, Thomson BA (2004) Collisional cooling of large ions in electrospray mass spectrometry. Anal Chem 76:1754–1760CrossRefPubMedGoogle Scholar
  23. 23.
    Giles K, Pringle SD, Worthington KR et al (2004) Applications of a travelling wave-based radio-frequency-only stacked ring ion guide. Rapid Commun Mass Spectrom 18:2401–2414CrossRefPubMedGoogle Scholar
  24. 24.
    Bush MF, Hall Z, Giles K et al (2010) Collision cross sections of proteins and their complexes: a calibration framework and database for gas-phase structural biology. Anal Chem 82:9557–9565CrossRefPubMedGoogle Scholar
  25. 25.
    Shvartsburg AA, Smith RD (2008) Fundamentals of traveling wave ion mobility spectrometry. Anal Chem 80:9689–9699PubMedCentralCrossRefPubMedGoogle Scholar
  26. 26.
    Thalassinos K, Grabenauer M, Slade SE et al (2009) Characterization of phosphorylated peptides using traveling wave-based and drift cell ion mobility mass spectrometry. Anal Chem 81:248–254CrossRefPubMedGoogle Scholar
  27. 27.
    Shvartsburg AA, Jarrold MF (1996) An exact hard-spheres scattering model for the mobilities of polyatomic ions. Chem Phys Lett 261:86–91CrossRefGoogle Scholar
  28. 28.
    Jarrold MF (1999) Unfolding, refolding, and hydration of proteins in the gas phase. Acc Chem Res 32:360–367CrossRefGoogle Scholar
  29. 29.
    Mack E (1925) Average cross-sectional areas of molecules by gaseous diffusion measurements. J Am Chem Soc 47:2468–2482CrossRefGoogle Scholar
  30. 30.
    Bleiholder C, Wyttenbach T, Bowers MT (2011) A novel projection approximation algorithm for the fast and accurate computation of molecular collision cross sections (i). Method. Int J Mass Spectrom 308:1–10CrossRefGoogle Scholar
  31. 31.
    Bleiholder C, Contreras S, Do TD et al (2013) A novel projection approximation algorithm for the fast and accurate computation of molecular collision cross sections (ii). Model parameterization and definition of empirical shape factors for proteins. Int J Mass Spectrom 345–347:89–96CrossRefGoogle Scholar
  32. 32.
    Bleiholder C, Contreras S, Bowers MT (2013) A novel projection approximation algorithm for the fast and accurate computation of molecular collision cross sections (iv). Application to polypeptides. Int J Mass Spectrom 354–355:275–280CrossRefGoogle Scholar
  33. 33.
    Anderson SE, Bleiholder C, Brocker ER et al (2012) A novel projection approximation algorithm for the fast and accurate computation of molecular collision cross sections (iii): Application to supramolecular coordination-driven assemblies with complex shapes. Int J Mass Spectrom 330–332:78–84CrossRefGoogle Scholar
  34. 34.
    Campuzano I, Giles K (2011) Nanospray ion mobility mass spectrometry of selected high mass species. In: Toms SA, Weil RJ (eds) Nanoproteomics, vol 790, Methods in molecular biology. Humana Press, Totowa, NJ, pp 57–70. doi: 10.1007/978-1-61779-319-6_5 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular BiologyUniversity of LeedsLeedsUK
  2. 2.Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular BiologyUniversity of LeedsLeedsUK

Personalised recommendations