Hydrogen rearrangement to and from radical z fragments in electron capture dissociation of peptides

  • Mikhail M. Savitski
  • Frank Kjeldsen
  • Michael L. Nielsen
  • Roman A. Zubarev


Hydrogen rearrangement is an important process in radical chemistry. A high degree of H· rearrangement to and from ionic fragments (combined occurrence frequency 47% compared with that of ) is confirmed in analysis of 15,000 tandem mass spectra of tryptic peptides obtained with electron capture dissociation (ECD), including previously unreported double H· losses. Consistent with the radical character of H· abstraction, the residue determining the formation rate of z′=z·+H· species is found to be the N-terminal residue in species. The size of the complementary cm fragment turned out to be another important factor, with z′ species dominating over z· ions for m ≤ 6. The H· atom was found to be abstracted from the side chains as well as from α-carbon groups of residues composing the c′ species, with Gln and His in the c′ fragment promoting H· donation and Asp and Ala opposing it. Ab initio calculations of formation energies of ·A radicals (A is an amino acid) confirmed that the main driving force for H· abstraction by z· is the process exothermicity. No valid correlation was found between the N-Cα bond strength and the frequency of this bond cleavage, indicating that other factors than thermochemistry are responsible for directing the site of ECD cleavage. Understanding hydrogen attachment to and loss from ECD fragments should facilitate automatic interpretation ECD mass spectra in protein identification and characterization, including de novo sequencing.


  1. 1.
    Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R. Direct Analysis of Protein Complexes Using Mass Spectrometry. Nat. Biotechnol. 1999, 17, 676–682.CrossRefGoogle Scholar
  2. 2.
    Pandey, A.; Mann, M. Proteomics to Study Genes and Genomes. Nature. 2000, 405, 837–846.CrossRefGoogle Scholar
  3. 3.
    Aebersold, R.; Mann, M. Mass Spectrometry-Based Proteomics. Nature. 2003, 422, 198–207.CrossRefGoogle Scholar
  4. 4.
    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
  5. 5.
    Nielsen, M. L.; Savitski, M. M.; Zubarev, R. A. Improving Protein Identification Using Complementary Fragmentation Techniques in Fourier Transform Mass Spectrometry. Mol. Cell. Proteomics. 2005, 4, 835–845.CrossRefGoogle Scholar
  6. 6.
    Savitski, M. M.; Nielsen, M. L.; Kjeldsen, F.; Zubarev, R. A. Proteomics-Grade de Novo Sequencing Approach. J. Proteome Res. 2005, 4, 2348–2354.CrossRefGoogle Scholar
  7. 7.
    Huang, Y. Y.; Triscari, J. M.; Tseng, G. C.; Pasa-Tolic, L.; Lipton, M. S.; Smith, R. D.; Wysocki, V. H. Statistical Characterization of the Charge State and Residue Dependence of Low-Energy CID Peptide Dissociation Patterns. Anal. Chem. 2005, 77, 5800–5813.CrossRefGoogle Scholar
  8. 8.
    O’Connor, P. B.; Lin, C.; Cournoyer, J. J.; Pittman, J. L.; Belyayev, M.; Budnik, B. A. Long-Lived Electron Capture Dissociation Product Ions Experience Radical Migration via Hydrogen Abstraction. J. Am. Soc. Mass Spectrom. 2006, 17, 576–585.CrossRefGoogle Scholar
  9. 9.
    Adams, C. M.; Kjeldsen, F.; Zubarev, R. A.; Budnik, B. A.; Haselmann, K. F. Electron Capture Dissociation Distinguishes a Single D-Amino Acid in a Protein and Probes the Tertiary Structure. J. Am. Soc. Mass Spectrom. 2004, 15, 1087–1098.CrossRefGoogle Scholar
  10. 10.
    Kjeldsen, F.; Savitski, M. M.; Adams, C. M.; Zubarev, R. A. Localization of Positive Charges in Gas-Phase Polypeptide Polycations by Tandem Mass Spectrometry. Int. J. Mass Spectrom. 2006, 252, 204–212.CrossRefGoogle Scholar
  11. 11.
    Savitski, M. M.; Kjeldsen, F.; Nielsen, M. L.; Zubarev, R. A. Complementary Sequence Preferences of Electron Capture Dissociation and Vibrational Excitation in Fragmentation of Polypeptide Polycations. Angew. Chem. Int. Ed. 2006, 45, 5301–5303.CrossRefGoogle Scholar
  12. 12.
    Zhang, R. Q.; Huang, J. H.; Bu, Y. X.; Han, K.; Lee, S. T.; He, G. Z. An Effective Scheme for Selecting Basis Sets for Ab Initio Calculations. Chin. Acad. J. 2000, 43, 375–388.Google Scholar
  13. 13.
    Dolgounitcheva, O.; Zakrzewski, V. G.; Ortiz, J. V. Structures and Electron Detachment Energies of Uracil Anions. Chem. Phys. Lett. 1999, 307, 220–226.CrossRefGoogle Scholar
  14. 14.
    Cooper, H. J.; Hudgins, R. R.; Hakansson, K.; Marshall, A. G. Secondary Fragmentation of Linear Peptides in Electron Capture Dissociation. Int. J. Mass Spectrom. 2003, 228, 723–728.CrossRefGoogle Scholar
  15. 15.
    Mirgorodskaya, E.; Roepstorff, P.; Zubarev, R. A. Localization of O-Glycosylation Sites in Peptides by Electron Capture Dissociation in a Fourier Transform Mass Spectrometer. Anal. Chem. 1999, 71, 4431–4436.CrossRefGoogle Scholar
  16. 16.
    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. 2000, 72, 563–573.CrossRefGoogle Scholar
  17. 17.
    Horn, D. M.; Zubarev, R. A.; McLafferty, F. W. Automated de Novo Sequencing of Proteins by Tandem High-Resolution Mass Spectrometry. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 10313–10317.CrossRefGoogle Scholar
  18. 18.
    Leymarie, N.; Costello, C. E.; O’Connor, P. B. Electron Capture Dissociation Initiates a Free Radical Reaction Cascade. J. Am. Chem. Soc. 2003, 125, 8949–8958.CrossRefGoogle Scholar
  19. 19.
    Turecek, F. N—C-α Bond Dissociation Energies and Kinetics in Amide and Peptide Radicals: Is the Dissociation a Nonergodic Process? J. Am. Chem. Soc. 2003, 125, 5954–5963.CrossRefGoogle Scholar
  20. 20.
    Turecek, F.; Syrstad, E. A. Mechanism and Energetics of Intramolecular Hydrogen Transfer in Amide and Peptide Radicals and Cation Radicals. J. Am. Chem. Soc. 2003, 125, 3353–3369.CrossRefGoogle Scholar
  21. 21.
    Zubarev, R. A.; Kruger, N. A.; Fridriksson, E. K.; Lewis, M. A.; Horn, D. M.; Carpenter, B. K.; McLafferty, F. W. Electron Capture Dissociation of Gaseous Multiply-Charged Proteins is Favored at Disulfide Bonds and Other Sites of High Hydrogen Atom Affinity. J. Am. Chem. Soc. 1999, 121, 2857–2862.CrossRefGoogle Scholar
  22. 22.
    Rauk, A.; Yu, D.; Armstrong, D. A. Toward Site Specificity of Oxidative Damage in Proteins: C—H and C—C Bond Dissociation Energies and Reduction Potentials of the Radicals of Alanine, Serine, and Threonine Residues—An ab Initio Study. J. Am. Chem. Soc. 1997, 119, 208–217.CrossRefGoogle Scholar
  23. 23.
    Rauk, A.; Yu, D.; Taylor, J.; Shustov, G. V.; Block, D. A.; Armstrong, D. A. Effects of structure on C-α—H Bond Enthalpies of Amino Acid Residues: Relevance to H Transfers in Enzyme Mechanisms and in Protein Oxidation. Biochemistry. 1999, 38, 9089–9096.CrossRefGoogle Scholar
  24. 24.
    McLafferty, F. W.; Turecek, F. Interpretation of Mass Spectra. Sausalito, CA: University Science Books, 1993.Google Scholar

Copyright information

© American Society for Mass Spectrometry 2007

Authors and Affiliations

  • Mikhail M. Savitski
    • 1
  • Frank Kjeldsen
    • 1
  • Michael L. Nielsen
    • 1
  • Roman A. Zubarev
    • 1
  1. 1.Laboratory for Biological and Medical Mass SpectrometryUppsala UniversityUppsalaSweden

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