Native electrospray and electron-capture dissociation in FTICR mass spectrometry provide top-down sequencing of a protein component in an intact protein assembly

  • Hao Zhang
  • Weidong Cui
  • Jianzhong Wen
  • Robert E. Blankenship
  • Michael L. Gross
Short Comunication


The intact yeast alcohol dehydrogenase (ADH) tetramer of 147 kDa was introduced into a FTICR mass spectrometer by native electrospray. Electron capture dissociation of the entire 23+ to 27+ charge state distribution produced the expected charge-reduced ions and, more unexpectedly, 39 c-type peptide fragments that identified N-terminus acetylation and the first 55 amino acids. The results are in accord with the crystal structure of yeast ADH, which shows that the C-terminus is buried at the assembly interface, whereas the N-terminus is exposed, allowing ECD to occur. This remarkable observation shows promise that a top-down approach for intact protein assemblies will be effective for characterizing their components, inferring their interfaces, and obtaining both proteomics and structural biology information in one experiment.


  1. 1.
    Benesch, J. L.; Ruotolo, B. T.; Simmons, D. A.; Robinson, C. V. Protein Complexes in the Gas Phase: Technology for Structural Genomics and Proteomics. Chem. Rev. 2007, 107(8), 3544–3567.CrossRefGoogle Scholar
  2. 2.
    Robinson, C. V.; Sali, A.; Baumeister, W. The Molecular Sociology of the Cell. Nature 2007, 450, 973–982.CrossRefGoogle Scholar
  3. 3.
    Benesch, J. L. Collisional Activation of Protein Complexes: Picking up the Pieces. J. Am. Soc. Mass Spectrom. 2009, 20, 341–348.CrossRefGoogle Scholar
  4. 4.
    Sharon, M. How Far Can We Go with Structural Mass Spectrometry of Protein Complexes? J. Am. Soc. Mass Spectrom. 2010, 21, 487–500.CrossRefGoogle Scholar
  5. 5.
    van Duijn, E. Current Limitations in Native Mass Spectrometry Based Structural Biology. J. Am. Soc. Mass Spectrom. 2010, 21, 971–978.CrossRefGoogle Scholar
  6. 6.
    Uetrecht, C.; Versluis, C.; Watts, N. R.; Roos, W. H.; Wuite, G. J.; Wingfield, P. T.; Steven, A. C.; Heck, A. J. High-Resolution Mass Spectrometry of Viral Assemblies: Molecular Composition and Stability of Dimorphic Hepatitis B Virus Capsids. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 9216–9220.CrossRefGoogle Scholar
  7. 7.
    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. 2001, 73, 4647–4661.CrossRefGoogle Scholar
  8. 8.
    Geels, R. B.; van der Vies, S. M.; Heck, A. J.; Heeren, R. M. Electron Capture Dissociation as Structural Probe for Noncovalent Gas-Phase Protein Assemblies. Anal. Chem. 2006, 78, 7191–7196.CrossRefGoogle Scholar
  9. 9.
    El-Faramawy, A.; Guo, Y.; Verkerk, U.; Thomson, B. A.; Siu, M. Evaluation of IR Multi photon Dissociation as a Method for High Mass Protein Clean up. Proceedings of the 56th ASMS Conference on Mass Spectrometry, Denver, CO, 2008.Google Scholar
  10. 10.
    Jones, C. M.; Beardsley, R. L.; Galhena, A. S.; Dagan, S.; Cheng, G.; Wysocki, V. H. Symmetrical Gas-Phase Dissociation of Noncovalent Protein Complexes Via Surface Collisions. J. Am. Chem. Soc. 2006, 128, 15044–15045.CrossRefGoogle Scholar
  11. 11.
    Benesch, J. L.; Ruotolo, B. T.; Sobott, F.; Wildgoose, J.; Gilbert, A.; Bateman, R.; Robinson, C. V. Quadrupole-Time-of-Flight Mass Spectrometer Modified for Higher-Energy Dissociation Reduces Protein Assemblies to Peptide Fragments. Anal. Chem. 2009, 81, 1270–1274.CrossRefGoogle Scholar
  12. 12.
    Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. Electron Capture Dissociation of Multiply Charged Protein Cations. A Nonergodic Process. J. Am. Chem. Soc. 1998, 120, 3265–3266.CrossRefGoogle Scholar
  13. 13.
    Han, X.; Jin, M.; Breuker, K.; McLafferty, F. W. Extending Top-Down Mass Spectrometry to Proteins with Masses Greater than 200 Kilodaltons. Science 2006, 314, 109–112.CrossRefGoogle Scholar
  14. 14.
    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. 2006, 128(45), 14432–14433.CrossRefGoogle Scholar
  15. 15.
    Guan, S.; Burlingame, A. L. High Mass Selectivity for Top-Down Proteomics by Application of SWIFT Technology. J. Am. Soc. Mass Spectrom. 2010, 21(3), 455–459.CrossRefGoogle Scholar
  16. 16.
    Powers, E. T.; Powers, D. L. A Perspective on Mechanisms of Protein Tetramer Formation. Biophys. J. 2003, 85, 3587–3599.CrossRefGoogle Scholar
  17. 17.
    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. 2002, 15(3), 215–223.CrossRefGoogle Scholar
  18. 18.
    Mortz, E.; O’Connor, C.; P. B.; Roepstorff, P.; Kelleher, N. L.; Wood, T. D.; McLafferty, F. W.; Mann, M. Sequence Tag Identification of Intact Proteins by Matching Tandem Mass Spectral Data Against Sequence Data Bases. Proc. Natl. Acad. Sci. U.S.A. 1996, 93(16), 8264–8267.CrossRefGoogle Scholar
  19. 19.
    Zhou, M.; Robinson, C. V. When Proteomics Meets Structural Biology. Trends Biochem Sci. doi:10.1016/j.tibs. 2010.04.007.Google Scholar

Copyright information

© American Society for Mass Spectrometry. Published by Elsevier Inc 2010

Authors and Affiliations

  • Hao Zhang
    • 1
  • Weidong Cui
    • 1
  • Jianzhong Wen
    • 1
  • Robert E. Blankenship
    • 1
  • Michael L. Gross
    • 1
  1. 1.Department of ChemistryWashington University in St. LouisSt. LouisUSA

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