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ETD Allows for Native Surface Mapping of a 150 kDa Noncovalent Complex on a Commercial Q-TWIMS-TOF Instrument

  • Research Article
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Journal of The American Society for Mass Spectrometry

Abstract

Top-down approaches for the characterization of intact proteins and macromolecular complexes are becoming increasingly popular, since they potentially simplify and speed up the assignment process. Here we demonstrate how, on a commercially available Q-TWIMS-TOF instrument, we performed top-down ETD of the native form of tetrameric alcohol dehydrogenase. We achieved good sequence coverage throughout the first 81 N-terminal amino acids of ADH, with the exception of a loop located on the inside of the protein. This is in agreement with the exposed parts of the natively folded protein according to the crystal structure. Choosing the right precursor charge state and applying supplemental activation were found to be key to obtaining a high ETD fragmentation efficiency. Finally, we briefly discuss opportunities to further increase the performance of ETD based on our results.

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References

  1. Rose, R.J., Damoc, E., Denisov, E., Makarov, A., Heck, A.J.: High-sensitivity Orbitrap mass analysis of intact macromolecular assemblies. Nat. Methods 9, 1084–1086 (2012)

    Article  CAS  Google Scholar 

  2. Tran, J.C., Zamdborg, L., Ahlf, D.R., Lee, J.E., Catherman, A.D., Durbin, K.R., Tipton, J.D., Vellaichamy, A., Kellie, J.F., Li, M., Wu, C., Sweet, S.M., Early, B.P., Siuti, N., LeDuc, R.D., Compton, P.D., Thomas, P.M., Kelleher, N.L.: Mapping intact protein isoforms in discovery mode using top-down proteomics. Nature 480, 254–258 (2011)

    Article  CAS  Google Scholar 

  3. Ruotolo, B.T., Benesch, J.L.P., Sandercock, A.M., Hyung, S.J., Robinson, C.V.: Ion mobility-mass spectrometry analysis of large protein complexes. Nat. Protoc. 3, 1139–1152 (2008)

    Article  CAS  Google Scholar 

  4. Kelleher, N.L., Lin, H.Y., Valaskovic, G.A., Aaserud, D.J., Fridriksson, E.K., McLafferty, F.: Top down versus bottom up protein characterization by tandem high-resolution mass spectrometry. J. Am. Chem. Soc. 121, 806–812 (1999)

    Google Scholar 

  5. Siuti, N., Kelleher, N.L.: Decoding protein modifications using top-down mass spectrometry. Nat. Methods 4, 817–821 (2007)

    Article  CAS  Google Scholar 

  6. Han, X., Jin, M., Breuker, K., McLafferty, F.W.: Extending top-down mass spectrometry to proteins with masses greater than 200 kilodaltons. Science 314, 109–112 (2006)

    Article  CAS  Google Scholar 

  7. Fornelli, L., Damoc, E., Thomas, P.M., Kelleher, N.L., Aizikov, K., Denisov, E., Makarov, A., Tsybin, Y.O.: Analysis of intact monoclonal antibody IgG1 by electron transfer dissociation Orbitrap FTMS. Mol. Cell. Proteom. 11, 1758–1767 (2012)

    Article  Google Scholar 

  8. Whitelegge, J.: Intact protein mass spectrometry and top-down proteomics. Expert Rev. Proteom. 10, 127–129 (2013)

    Article  CAS  Google Scholar 

  9. Konijnenberg, A., Butterer, A., Sobott, F.: Native ion mobility-mass spectrometry and related methods in structural biology. Biochim. Biophys. Acta. 1834, 1239–1256 (2013).

    Google Scholar 

  10. Tian, Z., Tolic, N., Zhao, R., Moore, R.J., Hengel, S.M., Robinson, E.W., Stenoien, D.L., Wu, S., Smith, R.D., Pasa-Tolic, L.: Enhanced top-down characterization of histone post-translational modifications. Genome Biol. 13, R86 (2012)

    Article  CAS  Google Scholar 

  11. Edwards, R.L., Griffiths, P., Bunch, J., Cooper, H.J.: Top-down proteomics and direct surface sampling of neonatal dried blood spots: diagnosis of unknown hemoglobin variants. J. Am. Soc. Mass Spectrom. 23, 1921–1930 (2012)

    Article  CAS  Google Scholar 

  12. Heck, A.J.: Native mass spectrometry: a bridge between interactomics and structural biology. Nat. Methods 5, 927–933 (2008)

    Article  CAS  Google Scholar 

  13. Hilton, G.R., Benesch, J.L.: Two decades of studying non-covalent biomolecular assemblies by means of electrospray ionization mass spectrometry. J. R. Soc. Interface 9, 801–816 (2012)

    Article  CAS  Google Scholar 

  14. Zhong, Y., Hyung, S.J., Ruotolo, B.T.: Ion mobility-mass spectrometry for structural proteomics. Expert Rev. Proteom. 9, 47–58 (2012)

    Article  CAS  Google Scholar 

  15. Barrera, N.P., Di Bartolo, N., Booth, P.J., Robinson, C.V.: Micelles protect membrane complexes from solution to vacuum. Science 321, 243–246 (2008)

    Article  CAS  Google Scholar 

  16. Morton, V.L., Stockley, P.G., Stonehouse, N.J., Ashcroft, A.E.: Insights into virus capsid assembly from noncovalent mass spectrometry. Mass Spectrom. Rev. 27, 575–595 (2008)

    Article  Google Scholar 

  17. Benesch, J.L.P.: Collisional activation of protein complexes: picking up the pieces. J. Am. Soc. Mass Spectrom. 20, 341–348 (2009)

    Article  CAS  Google Scholar 

  18. Zubarev, R.A., Horn, D.M., Fridriksson, E.K., Kelleher, N.L., Kruger, N.A., Lewis, M.A., Carpenter, B.K., McLafferty, F.W.: Electron capture dissociation for structural characterization of multiply charged protein cations. Anal. Chem. 72, 563–573 (2000)

    Article  CAS  Google Scholar 

  19. Zubarev, R.A., Kelleher, N.L., McLafferty, F.: Electron capture dissociation of multiply charged protein cations. A nonergodic process. J. Am. Chem. Soc. 120, 3265–3266 (1998)

    Google Scholar 

  20. Tsybin, Y.O., Fornelli, L., Stoermer, C., Luebeck, M., Parra, J., Nallet, S., Wurm, F.M., Hartmer, R.: Structural analysis of intact monoclonal antibodies by electron transfer dissociation mass spectrometry. Anal. Chem. 83, 8919–8927 (2011)

    Article  CAS  Google Scholar 

  21. Syka, J.E., Coon, J.J., Schroeder, M.J., Shabanowitz, J., Hunt, D.F.: Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc. Natl. Acad. Sci. U. S. A. 101, 9528–9533 (2004)

    Article  CAS  Google Scholar 

  22. Coon, J.J., Ueberheide, B., Syka, J.E., Dryhurst, D.D., Ausio, J., Shabanowitz, J., Hunt, D.F.: Protein identification using sequential ion/ion reactions and tandem mass spectrometry. Proc. Natl. Acad. Sci. U. S. A. 102, 9463–9468 (2005)

    Article  CAS  Google Scholar 

  23. Sobott, F., Watt, S.J., Smith, J., Edelmann, M.J., Kramer, H.B., Kessler, B.M.: Comparison of CID versus ETD based MS/MS fragmentation for the analysis of protein ubiquitination. J. Am. Soc. Mass Spectrom. 20, 1652–1659 (2009)

    Article  CAS  Google Scholar 

  24. Garcia, B.A., Shabanowitz, J., Hunt, D.F.: Characterization of histones and their post-translational modifications by mass spectrometry. Curr. Opin. Chem. Biol. 11, 66–73 (2007)

    Article  CAS  Google Scholar 

  25. Zhang, H., Cui, W., Wen, J., Blankenship, R.E., Gross, M.L.: Native electrospray and electron-capture dissociation in FTICR mass spectrometry provide top-down sequencing of a protein component in an intact protein assembly. J. Am. Soc. Mass Spectrom. 21, 1966–1968 (2010)

    Article  CAS  Google Scholar 

  26. Xie, Y., Zhang, J., Yin, S., Loo, J.A.: Top-Down ESI-ECD-FT-ICR mass spectrometry localizes noncovalent protein-ligand binding sites. J. Am. Chem. Soc. 128, 14432–14433 (2006)

    Article  CAS  Google Scholar 

  27. Zhang, H., Cui, W., Wen, J., Blankenship, R.E., Gross, M.L.: Native electrospray and electron-capture dissociation FTICR mass spectrometry for top-down studies of protein assemblies. Anal. Chem. 83, 5598–5606 (2011)

    Article  CAS  Google Scholar 

  28. Jones, L.M., Zhang, H., Cui, W., Kumar, S., Sperry, J.B., Carroll, J.A., Gross, M.L.: Complementary MS methods assist conformational characterization of antibodies with altered S–S bonding networks. J. Am. Soc. Mass Spectrom. 24, 835–845 (2013)

    Article  CAS  Google Scholar 

  29. Geels, R.B.J., van der Vies, S.M., Heck, A.J., Heeren, R.M.A.: Electron capture dissociation as structural probe for noncovalent gas-phase protein assemblies. Anal. Chem. 78, 7191–7196 (2006)

    Article  CAS  Google Scholar 

  30. Williams, J.P., Brown, J.M., Campuzano, I., Sadler, P.J.: Identifying drug metallation sites on peptides using electron transfer dissociation (ETD), collision induced dissociation (CID) and ion mobility-mass spectrometry (IM-MS). Chem. Commun. 46, 5458–5460 (2010)

    Article  CAS  Google Scholar 

  31. Rand, K.D., Pringle, S.D., Morris, M., Engen, J.R., Brown, J.M.: ETD in a traveling wave ion guide at tuned z-spray ion source conditions allows for site-specific hydrogen/deuterium exchange measurements. J. Am. Soc. Mass Spectrom. 22, 1784–1793 (2011)

    Article  CAS  Google Scholar 

  32. McLuckey, S.A., Stephenson Jr., J.L.: Ion/ion chemistry of high-mass multiply charged ions. Mass Spectrom. Rev. 17, 369–407 (1998)

    Article  CAS  Google Scholar 

  33. Powers, E.T., Powers, D.L.: A perspective on mechanisms of protein tetramer formation. Biophys. J. 85, 3587–3599 (2003)

    Article  CAS  Google Scholar 

  34. Casadio, R., Martelli, P.L., Giordano, A., Rossi, M., Raia, C.A.: A low-resolution 3D model of the tetrameric alcohol dehydrogenase from Sulfolobus solfataricus. Protein Eng. 15, 215–223 (2002)

    Article  CAS  Google Scholar 

  35. 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. 261, 1–12 (2007)

    Article  CAS  Google Scholar 

  36. Yasara Dynamics. Available at: www.yasara.org. Accessed 4 Aug 2013

  37. Wang, J., Cieplak, P., Kollman, P.A.: How well does a restrained electrostatic potential (RESP) model perform in calculating conformational energies of organic and biological molecules? J. Comput. Chem. 21, 1049–1074 (2000)

    Article  CAS  Google Scholar 

  38. Rozman, M., Gaskell, S.J.: Charge state dependent top-down characterisation using electron transfer dissociation. Rapid Commun. Mass Spectrom. 26, 282–286 (2012)

    Article  CAS  Google Scholar 

  39. Good, D.M., Wirtala, M., McAlister, G.C., Coon, J.J.: Performance characteristics of electron transfer dissociation mass spectrometry. Mol. Cell. Proteom. 6, 1942–1951 (2007)

    Article  CAS  Google Scholar 

  40. Liu, J., McLuckey, S.A.: Electron transfer dissociation: effects of cation charge state on product partitioning in ion/ion electron transfer to multiply protonated polypeptides. Int. J. Mass Spectrom 330/332, 174–181 (2012)

    Article  Google Scholar 

  41. Hall, Z., Robinson, C.V.: Do charge state signatures guarantee protein conformations? J. Am. Soc. Mass Spectrom. 23, 1161–1168 (2012)

    Article  CAS  Google Scholar 

  42. Hamdy, O.M., Julian, R.R.: Reflections on charge state distributions, protein structure, and the mystical mechanism of electrospray ionization. J. Am. Soc. Mass Spectrom. 23, 1–6 (2012)

    Article  CAS  Google Scholar 

  43. Lomeli, S.H., Peng, I.X., Yin, S., Loo, R.R., Loo, J.A.: New reagents for increasing ESI multiple charging of proteins and protein complexes. J. Am. Soc. Mass Spectrom. 21, 127–131 (2010)

    Article  CAS  Google Scholar 

  44. Lomeli, S.H., Yin, S., Loo, R.R., Loo, J.A.: Increasing charge while preserving noncovalent protein complexes for ESI-MS. J. Am. Soc. Mass Spectrom. 20, 593–596 (2008)

    Article  Google Scholar 

  45. Hogan Jr., C.J., Ogorzalek Loo, R.R., Loo, J.A., de la Mora, J.F.: Ion mobility-mass spectrometry of phosphorylase B ions generated with supercharging reagents but in charge-reducing buffer. Phys. Chem., Chem. Phys. 12, 13476–13483 (2010)

    Article  CAS  Google Scholar 

  46. Sterling, H.J., Daly, M.P., Feld, G.K., Thoren, K.L., Kintzer, A.F., Krantz, B.A., Williams, E.R.: Effects of supercharging reagents on noncovalent complex structure in electrospray ionization from aqueous solutions. J. Am. Soc. Mass Spectrom. 21, 1762–1774 (2010)

    Article  CAS  Google Scholar 

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Acknowledgments

F.L. acknowledges financial support in the form of a Ph.D. fellowship of the Research Foundation – Flanders (FWO), and F.S. is a Francqui Research Professor at UA. The Synapt G2 mass spectrometer is funded by a grant from the Hercules Foundation – Flanders. Financial support by the Flemish Institute for Technological Research (VITO) is gratefully acknowledged. The authors also thank the reviewers for their valuable comments.

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Authors and Affiliations

  1. Biomolecular and Analytical Mass Spectrometry group, Department of Chemistry, University of Antwerp, Groenenborgerlaan 171, 2020, Antwerpen, Belgium

    Frederik Lermyte, Albert Konijnenberg & Frank Sobott

  2. Center for Proteomics (CFP-CeProMa), University of Antwerp, Antwerpen, Belgium

    Frederik Lermyte, Dirk Valkenborg & Frank Sobott

  3. Waters Corporation, Atlas Park, Simonsway, Wythenshawe, Manchester, UK

    Jonathan P. Williams & Jeffery M. Brown

  4. Applied Bio and Molecular Systems, VITO, Mol, Belgium

    Dirk Valkenborg

  5. Interuniversity Institute for Biostatistics and Statistical Bioinformatics, Hasselt University, Diepenbeek, Belgium

    Dirk Valkenborg

Authors
  1. Frederik Lermyte
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  2. Albert Konijnenberg
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  3. Jonathan P. Williams
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  4. Jeffery M. Brown
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  5. Dirk Valkenborg
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  6. Frank Sobott
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Corresponding author

Correspondence to Frank Sobott.

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Frederik Lermyte and Albert Konijnenberg contributed equally to this work

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Lermyte, F., Konijnenberg, A., Williams, J.P. et al. ETD Allows for Native Surface Mapping of a 150 kDa Noncovalent Complex on a Commercial Q-TWIMS-TOF Instrument. J. Am. Soc. Mass Spectrom. 25, 343–350 (2014). https://doi.org/10.1007/s13361-013-0798-3

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  • DOI: https://doi.org/10.1007/s13361-013-0798-3

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