Skip to main content
SpringerLink
Log in

Traveling-wave ion mobility-mass spectrometry reveals additional mechanistic details in the stabilization of protein complex ions through tuned salt additives

  • Original Research
  • Published:
International Journal for Ion Mobility Spectrometry

Abstract

Ion mobility–mass spectrometry is often applied to the structural elucidation of multiprotein assemblies in cases where X-ray crystallography or NMR experiments have proved challenging. Such applications are growing steadily as we continue to probe regions of the proteome that are less-accessible to such high-resolution structural biology tools. Since ion mobility measures protein structure in the absence of bulk solvent, strategies designed to more-broadly stabilize native-like protein structures in the gas-phase would greatly enable the application of such measurements to challenging structural targets. Recently, we have begun investigating the ability of salt-based solution additives that remain bound to protein ions in the gas-phase to stabilize native-like protein structures. These experiments, which utilize collision induced unfolding and collision induced dissociation in a tandem mass spectrometry mode to measure protein stability, seek to develop a rank-order similar to the Hofmeister series that categorizes the general ability of different anions and cations to stabilize gas-phase protein structure. Here, we study magnesium chloride as a potential stabilizing additive for protein structures in vacuo, and find that the addition of this salt to solutions prior to nano-electrospray ionization dramatically enhances multiprotein complex structural stability in the gas-phase. Based on these experiments, we also refine the physical mechanism of cation-based protein complex ion stabilization by tracking the unfolding transitions experienced by cation-bound complexes. Upon comparison with unbound proteins, we find strong evidence that stabilizing cations act to tether protein complex structure. We conclude by putting the results reported here in context, and by projecting the future applications of this method.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Annesley TM (2003) Ion suppression in mass spectrometry. Clin Chem 49(7):1041–1044. doi:10.1373/49.7.1041

    Article  CAS  Google Scholar 

  2. Aquilina JA, Benesch JLP, Bateman OA, Slingsby C, Robinson CV (2003) Polydispersity of a mammalian chaperone: mass spectrometry reveals the population of oligomers in alpha B-crystallin. Proc Natl Acad Sci U S A 100(19):10611–10616. doi:10.1073/pnas.1932958100

    Article  CAS  Google Scholar 

  3. Badman ER, Myung S, Clemmer DE (2005) Evidence for unfolding and refolding of gas-phase cytochrome c ions in a Paul trap. Journal of the American Society for Mass Spectrometry 16(9):1493–1497. doi:10.1016/j.jasms.2005.04.013

    Article  CAS  Google Scholar 

  4. Bagal D, Kitova EN, Liu L, El-Hawiet A, Schnier PD, Klassen JS (2009) Gas phase stabilization of noncovalent protein complexes formed by electrospray ionization. Anal Chem 81(18):7801–7806. doi:10.1021/ac900611a

    Article  CAS  Google Scholar 

  5. Barrera NP, Isaacson SC, Zhou M, Bavro VN, Welch A, Schaedler TA, Seeger MA, Miguel RN, Korkhov VM, van Veen HW, Venter H, Walmsley AR, Tate CG, Robinson CV (2009) Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions. Nature Methods 6(8):585–U549. doi:10.1038/nmeth.1347

    Article  CAS  Google Scholar 

  6. Benesch JLP (2009) Collisional activation of protein complexes: picking up the pieces. Journal of the American Society for Mass Spectrometry 20(3):341–348. doi:10.1016/j.jasms.2008.11.014

    Article  CAS  Google Scholar 

  7. Benesch JLP, Ruotolo BT (2011) Mass spectrometry: come of age for structural and dynamical biology. Curr Opin Struct Biol 21(5):641–649. doi:10.1016/j.sbi.2011.08.002

    Article  CAS  Google Scholar 

  8. Benesch JLP, Ruotolo BT, Simmons DA, Robinson CV (2007) Protein complexes in the gas phase: technology for structural genomics and proteomics. Chem Rev 107(8):3544–3567. doi:10.1021/cr068289b

    Article  CAS  Google Scholar 

  9. Bohrer BC, Merenbloom SI, Koeniger SL, Hilderbrand AE, Clemmer DE (2008) Biomolecule analysis by ion mobility spectrometry. Annu Rev Anal Chem 1:293–327

    Article  CAS  Google Scholar 

  10. Breuker K, McLafferty FW (2008) Stepwise evolution of protein native structure with electrospray into the gas phase, 10(−12) to 10(2) S. Proc Natl Acad Sci U S A 105(47):18145–18152. doi:10.1073/pnas.0807005105

    Article  CAS  Google Scholar 

  11. Bush MF, Hall Z, Giles K, Hoyes J, Robinson CV, Ruotolo BT (2010) Collision cross sections of proteins and their complexes: a calibration framework and database for gas-phase structural biology. Anal Chem 82(22):9557–9565. doi:10.1021/ac1022953

    Article  CAS  Google Scholar 

  12. Carulla N, Zhou M, Giralt E, Robinson CV, Dobson CM (2010) Structure and intermolecular dynamics of aggregates populated during amyloid fibril formation studied by hydrogen/deuterium exchange. Accounts of Chemical Research 43(8):1072–1079. doi:10.1021/ar9002784

    Article  CAS  Google Scholar 

  13. Clemmer DE, Jarrold MF (1997) Ion mobility measurements and their applications to clusters and biomolecules. Journal of Mass Spectrometry 32(6):577–592. doi:10.1002/(sici)1096-9888(199706)32:6<577::aid-jms530>3.3.co;2-w

    Article  CAS  Google Scholar 

  14. Cole HL, Kalapothakis JMD, Bennett G, Barran PE, MacPhee CE (2010) Characterizing early aggregates formed by an amyloidogenic peptide by mass spectrometry. Angew Chem Int Ed 49(49):9448–9451. doi:10.1002/anie.201003373

    Article  CAS  Google Scholar 

  15. Ekeowa UI, Freeke J, Miranda E, Gooptu B, Bush MF, Perez J, Teckman J, Robinson CV, Lomas DA (2010) Defining the mechanism of polymerization in the serpinopathies. Proc Natl Acad Sci U S A 107(40):17146–17151. doi:10.1073/pnas.1004785107

    Article  CAS  Google Scholar 

  16. Freeke J, Bush MF, Robinson CV, Ruotolo BT (2012) Gas-phase protein assemblies: unfolding landscapes and preserving native-like structures using noncovalent adducts. Chem Phys Lett 524:1–9. doi:10.1016/j.cplett.2011.11.014

    Article  CAS  Google Scholar 

  17. Freeke J, Robinson CV, Ruotolo BT (2010) Residual counter ions can stabilise a large protein complex in the gas phase. International Journal of Mass Spectrometry 298(1–3):91–98. doi:10.1016/j.ijms.2009.08.001

    Article  CAS  Google Scholar 

  18. Giles K, Pringle SD, Worthington KR, Little D, Wildgoose JL, Bateman RH (2004) Applications of a travelling wave-based radio-frequencyonly stacked ring ion guide. Rapid Communications in Mass Spectrometry 18(20):2401–2414. doi:10.1002/rcm.1641

    Article  CAS  Google Scholar 

  19. Giles K, Williams JP, Campuzano I (2011) Enhancements in travelling wave ion mobility resolution. Rapid Communications in Mass Spectrometry 25(11):1559–1566. doi:10.1002/rcm.5013

    Article  CAS  Google Scholar 

  20. Han L, Hyung SJ, Ruotolo BT (2012) Bound cations significantly stabilize the structure of multiprotein complexes in the gas phase. Angew Chem Int Ed Engl. doi:10.1002/anie.201109127

  21. Han L, Hyung SJ, Ruotolo BT (2013) Dramatically stabilizing multiprotein complex structure in the absence of bulk water using tuned Hofmeister salts. Faraday Discussions

  22. Han LJ, Hyung SJ, Mayers JJS, Ruotolo BT (2011) Bound anions differentially stabilize multiprotein complexes in the absence of bulk solvent. J Am Chem Soc 133(29):11358–11367. doi:10.1021/ja203527a

    Article  CAS  Google Scholar 

  23. Heck AJR (2008) Native mass spectrometry: a bridge between interactomics and structural biology. Nature Methods 5(11):927–933. doi:10.1038/nmeth.1265

    Article  CAS  Google Scholar 

  24. Heck AJR, van den Heuvel RHH (2004) Investigation of intact protein complexes by mass spectrometry. Mass Spectrometry Reviews 23(5):368–389. doi:10.1002/mas.10081

    Article  CAS  Google Scholar 

  25. Hernandez H, Dziembowski A, Taverner T, Seraphin B, Robinson CV (2006) Subunit architecture of multimeric complexes isolated directly from cells. Embo Reports 7(6):605–610. doi:10.1038/sj.embor.7400702

    CAS  Google Scholar 

  26. Hernandez H, Makarova OV, Makarov EM, Morgner N, Muto Y, Krummel DP, Robinson CV (2009) Isoforms of U1-70 k control subunit dynamics in the human spliceosomal U1 snRNP. PLoS One 4(9):doi:e720210.1371/journal.pone.0007202

    Article  Google Scholar 

  27. Hernandez H, Robinson CV (2007) Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry. Nat Protoc 2(3):715–726. doi:10.1038/nprot.2007.73

    Article  CAS  Google Scholar 

  28. Hogan CJ, Ruotolo BT, Robinson CV, de la Mora JF (2011) Tandem differential mobility analysis-mass spectrometry reveals partial gas-phase collapse of the GroEL complex. Journal of Physical Chemistry B 115(13):3614–3621. doi:10.1021/jp109172k

    Article  CAS  Google Scholar 

  29. Hopper JTS, Oldham NJ (2009) Collision induced unfolding of protein ions in the gas phase studied by ion mobility-mass spectrometry: the effect of ligand binding on conformational stability. Journal of the American Society for Mass Spectrometry 20(10):1851–1858. doi:10.1016/j.jasms.2009.06.010

    Article  CAS  Google Scholar 

  30. Hyung SJ, Robinson CV, Ruotolo BT (2009) Gas-phase unfolding and disassembly reveals stability differences in ligand-bound multiprotein complexes. Chem Biol 16(4):382–390. doi:10.1016/j.chembiol.2009.02.008

    Article  CAS  Google Scholar 

  31. Hyung SJ, Ruotolo BT (2012) Integrating mass spectrometry of intact protein complexes into structural proteomics. Proteomics In press

  32. Jurchen JC, Williams ER (2003) Origin of asymmetric charge partitioning in the dissociation of gas-phase protein homodimers. J Am Chem Soc 125(9):2817–2826. doi:10.1021/ja0211508

    Article  CAS  Google Scholar 

  33. Jurneczko E, Barran PE (2011) How useful is ion mobility mass spectrometry for structural biology? The relationship between protein crystal structures and their collision cross sections in the gas phase. Analyst 136(1):20–28. doi:10.1039/c0an00373e

    Article  CAS  Google Scholar 

  34. Kaddis CS, Lomeli SH, Yin S, Berhane B, Apostol MI, Kickhoefer VA, Rome LH, Loo JA (2007) Sizing large proteins and protein complexes by electrospray ionization mass spectrometry and ion mobility. Journal of the American Society for Mass Spectrometry 18(7):1206–1216. doi:10.1016/j.jasms.2007.02.015

    Article  CAS  Google Scholar 

  35. Knapman TW, Morton VL, Stonehouse NJ, Stockley PG, Ashcroft AE (2010) Determining the topology of virus assembly intermediates using ion mobility spectrometry-mass spectrometry. Rapid Communications in Mass Spectrometry 24(20):3033–3042. doi:10.1002/rcm.4732

    Article  CAS  Google Scholar 

  36. McKay AR, Ruotolo BT, Ilag LL, Robinson CV (2006) Mass measurements of increased accuracy resolve heterogeneous populations of intact ribosomes. J Am Chem Soc 128(35):11433–11442. doi:10.1021/ja061468q

    Article  CAS  Google Scholar 

  37. Merenbloom SI, Flick TG, Daly MP, Williams ER (2011) Effects of select anions from the hofmeister series on the gas-phase conformations of protein ions measured with traveling-wave ion mobility spectrometry/mass spectrometry. Journal of the American Society for Mass Spectrometry pp 1–13

  38. Pagel K, Hyung SJ, Ruotolo BT, Robinson CV (2010) Alternate dissociation pathways identified in charge-reduced protein complex ions. Anal Chem 82(12):5363–5372. doi:10.1021/ac101121r

    Article  CAS  Google Scholar 

  39. Pierce KL, Premont RT, Lefkowitz RJ (2002) Seven-transmembrane receptors. Nat Rev Mol Cell Biol 3(9):639–650. doi:10.1038/nrm908

    Article  CAS  Google Scholar 

  40. Politis A, Park AY, Hyung SJ, Barsky D, Ruotolo BT, Robinson CV (2010) Integrating ion mobility mass spectrometry with molecular modelling to determine the architecture of multiprotein complexes. PLoS One 5(8):doi:e1208010.1371/journal.pone.0012080

    Article  Google Scholar 

  41. Pukala TL, Ruotolo BT, Zhou M, Politis A, Stefanescu R, Leary JA, Robinson CV (2009) Subunit architecture of multiprotein assemblies determined using restraints from gas-phase measurements. Structure 17(9):1235–1243. doi:10.1016/j.str.2009.07.013

    Article  CAS  Google Scholar 

  42. Robinson CV, Sali A, Baumeister W (2007) The molecular sociology of the cell. Nature 450(7172):973–982. doi:10.1038/nature06523

    Article  CAS  Google Scholar 

  43. Ruotolo BT, Benesch JLP, Sandercock AM, Hyung SJ, Robinson CV (2008) Ion mobility-mass spectrometry analysis of large protein complexes. Nat Protoc 3(7):1139–1152. doi:10.1038/nprot.2008.78

    Article  CAS  Google Scholar 

  44. Ruotolo BT, Giles K, Campuzano I, Sandercock AM, Bateman RH, Robinson CV (2005) Evidence for macromolecular protein rings in the absence of bulk water. Science 310(5754):1658–1661. doi:10.1126/science.1120177

    Article  CAS  Google Scholar 

  45. Ruotolo BT, Robinson CV (2006) Aspects of native proteins are retained in vacuum. Curr Opin Chem Biol 10(5):402–408. doi:10.1016/j.cbpa.2006.08.020

    Article  CAS  Google Scholar 

  46. Sali A, Glaeser R, Earnest T, Baumeister W (2003) From words to literature in structural proteomics. Nature 422(6928):216–225. doi:10.1038/nature01513

    Article  CAS  Google Scholar 

  47. Scarff CA, Patel VJ, Thalassinos K, Scrivens JH (2009) Probing hemoglobin structure by means of traveling-wave ion mobility mass spectrometry. Journal of the American Society for Mass Spectrometry 20(4):625–631. doi:10.1016/j.jasms.2008.11.023

    Article  CAS  Google Scholar 

  48. Scarff CA, Thalassinos K, Hilton GR, Scrivens JH (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 Communications in Mass Spectrometry 22(20):3297–3304. doi:10.1002/rcm.3737

    Article  CAS  Google Scholar 

  49. Sharon M, Robinson CV (2007) The role of mass Spectrometry in structure elucidation of dynamic protein complexes. Annu Rev Biochem 76:167–193. doi:10.1146/annurev.biochem.76.061005.090816

    Article  CAS  Google Scholar 

  50. Smith DP, Radford SE, Ashcroft AE (2010) Elongated oligomers in beta(2)-microglobulin amyloid assembly revealed by ion mobility spectrometry-mass spectrometry. Proc Natl Acad Sci U S A 107(15):6794–6798. doi:10.1073/pnas.0913046107

    Article  CAS  Google Scholar 

  51. Steven AC, Baumeister W (2008) The future is hybrid. J Struct Biol 163(3):186–195. doi:10.1016/j.jsb.2008.06.002

    Article  CAS  Google Scholar 

  52. Sun JX, Kitova EN, Klassen JS (2007) Method for stabilizing protein-ligand complexes in nanoelectrospray ionization mass spectrometry. Anal Chem 79(2):416–425. doi:10.1021/ac061109d

    Article  CAS  Google Scholar 

  53. Uetrecht C, Rose RJ, van Duijn E, Lorenzen K, Heck AJR (2010) Ion mobility mass spectrometry of proteins and protein assemblies. Chem Soc Rev 39(5):1633–1655. doi:10.1039/b914002f

    Article  CAS  Google Scholar 

  54. von Heijne G (2006) Membrane-protein topology. Nat Rev Mol Cell Biol 7(12):909–918. doi:10.1038/nrm2063

    Article  Google Scholar 

  55. Wyttenbach T, Bowers MT (2007) Intermolecular interactions in biomolecular systems examined by mass spectrometry. Annu Rev Phys Chem 58:511–533. doi:10.1146/annurev.physchem.58.032806.104515

    Article  CAS  Google Scholar 

  56. Zhong Y, Hyung S-J, Ruotolo BT (2012) Ion mobility–mass spectrometry for structural proteomics. Expert Review of Proteomics 9(1):47–58. doi:10.1586/epr.11.75

    Article  CAS  Google Scholar 

  57. Zhong YY, Hyung SJ, Ruotolo BT (2011) Characterizing the resolution and accuracy of a second-generation traveling-wave ion mobility separator for biomolecular ions. Analyst 136(17):3534–3541. doi:10.1039/c0an00987c

    Article  CAS  Google Scholar 

  58. Zhou M, Morgner N, Barrera NP, Politis A, Isaacson SC, Matak-Vinković D, Murata T, Bernal RA, Stock D, Robinson CV (2011) Mass spectrometry of intact V-Type ATPases reveals bound lipids and the effects of nucleotide binding. Science 334(6054):380–385. doi:10.1126/science.1210148

    Article  CAS  Google Scholar 

  59. Zhou M, Sandercock AM, Fraser CS, Ridlova G, Stephens E, Schenauer MR, Yokoi-Fong T, Barsky D, Leary JA, Hershey JW, Doudna JA, Robinson CV (2008) Mass spectrometry reveals modularity and a complete subunit interaction map of the eukaryotic translation factor eIF3. Proc Natl Acad Sci U S A 105(47):18139–18144. doi:10.1073/pnas.0801313105

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work is supported by the National Institutes of Health (1-R01-GM-095832-01) and by University of Michigan startup funds.

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Michigan, 930 N. University Ave., Ann Arbor, MI, 48108, USA

    Linjie Han & Brandon T. Ruotolo

Authors
  1. Linjie Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Brandon T. Ruotolo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Brandon T. Ruotolo.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Han, L., Ruotolo, B.T. Traveling-wave ion mobility-mass spectrometry reveals additional mechanistic details in the stabilization of protein complex ions through tuned salt additives. Int. J. Ion Mobil. Spec. 16, 41–50 (2013). https://doi.org/10.1007/s12127-013-0121-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12127-013-0121-9

Keywords

Search

Navigation