The effect of radical trap moieties on electron capture dissociation spectra of substance P

  • Marina A. Belyayev
  • Jason J. Cournoyer
  • Cheng Lin
  • Peter B. O’Connor
Article

Abstract

To further test the hypothesis that electron capture dissociation (ECD) involves long-lived radical intermediates and radical migration occurs within these intermediates before fragmentation, radical trap moieties were attached to peptides with the assumption that they would reduce fragmentation by decreasing the mobility of the radical. Coumarin labels were chosen for the radical traps, and unlabeled, singly-labeled, and doubly-labeled Substance P were analyzed by ECD. The results demonstrated a correlation between the number and position of tags on the peptide and the intensity of side-chain cleavages observed, as well as an inverse correlation between the number of tags on the peptide and the intensity of backbone cleavages. Addition of radical traps to the peptide inhibits backbone cleavages, suggesting that either radical mobility is required for these cleavages, or new noncovalent interactions prevent separation of backbone cleavage fragments. The enhancement of side-chain cleavages and the observation of new side-chain cleavages associated with aromatic groups suggest that the gas-phase conformation of this peptide is substantially distorted from untagged Substance P and involves previously unobserved interactions between the coumarin tags and the phenylalanine residues. Furthermore, the use of a double resonance (DR)-ECD experiment showed that these side-chain losses are all products of long-lived radical intermediate species, which suggests that steric hindrance prevents the coumarin-localized radical from interacting with the backbone while simultaneously increasing the radical rearrangements with the side chains.

References

  1. 1.
    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
  2. 2.
    Zubarev, R. A. Reactions of Polypeptide Ions with Electrons in the Gas Phase. Mass Spectrom. Rev. 2003, 22, 57–77.CrossRefGoogle Scholar
  3. 3.
    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
  4. 4.
    Kelleher, N. L.; Zubarev, R. A.; Bush, K.; Furie, B.; Furie, B. C.; McLafferty, F. W.; Walsh, C. T. Localization of Labile Post-translational Modifications by Electron Capture Dissociation: The Case of γ-Carboxyglutamic Acid. Anal. Chem. 1999, 71, 4250–4253.CrossRefGoogle Scholar
  5. 5.
    Stensballe, A.; Jensen, O. N.; Olsen, J. V.; Haselmann, K. F.; Zubarev, R. A. Electron Capture Dissociation of Singly and Multiply Phosphorylated Peptides. Rapid Commun. Mass Spectrom. 2000, 14, 1793–1800.CrossRefGoogle Scholar
  6. 6.
    Shi, S. D. H.; Hemling, M. E.; Carr, S. A.; Horn, D. M.; Lindh, I.; McLafferty, F. W. Phosphopeptide/Phosphoprotein Mapping by Electron Capture Dissociation Mass Spectrometry. Anal. Chem. 2001, 73, 19–22.CrossRefGoogle Scholar
  7. 7.
    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
  8. 8.
    Hakansson, K.; Cooper, H. J.; Emmett, M. R.; Costello, C. E.; Marshall, A. G.; Nilsson, C. L. Electron Capture Dissociation and Infrared Multiphoton Dissociation MS/MS of an N-Glycosylated Tryptic Peptide to Yield Complementary Sequence Information. Anal. Chem. 2001, 73, 4530–4536.CrossRefGoogle Scholar
  9. 9.
    Patriksson, A.; Adams, C.; Kjeldsen, F.; Raber, J.; van der Spoel, D.; Zubarev, R. A. Prediction of N-C-α Bond Cleavage Frequencies in Electron Capture Dissociation of Trp-Cage Dications by Force-Field Molecular Dynamics Simulations. Int. J. Mass Spectrom. Ion Processes 2006, 248, 124–135.Google Scholar
  10. 10.
    Oh, H.; Breuker, K.; Sze, S. K.; Ge, Y.; Carpenter, B. K.; McLafferty, F. W. Secondary and Tertiary Structures of Gaseous Protein Ions Characterized by Electron Capture Dissociation Mass Spectrometry and Photofragment Spectroscopy. Proc. Nat. Acad. Sci. U.S.A. 2002, 99, 15863–15868.CrossRefGoogle Scholar
  11. 11.
    Breuker, K.; Oh, H.; Horn, D. M.; Cerda, B. A.; McLafferty, F. W. Detailed Unfolding and Folding of Gaseous Ubiquitin Ions Characterized by Electron Capture Dissociation. J. Am. Chem. Soc. 2002, 124, 6407–6420.CrossRefGoogle Scholar
  12. 12.
    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
  13. 13.
    Leymarie, N.; Berg, E. A.; McComb, M. E.; O’Connor, P. B.; Grogan, J.; Oppenheim, F. G.; Costello, C. E. Tandem Mass Spectrometry for Structural Characterization of Proline-Rich Proteins: Application to Salivary PRP-3. Anal. Chem. 2002, 74, 4124–4132.CrossRefGoogle Scholar
  14. 14.
    Syrstad, E. A.; Turecek, F. Toward a General Mechanism of Electron Capture Dissociation. J. Am. Soc. Mass Spectrom. 2005, 16, 208–224.CrossRefGoogle Scholar
  15. 15.
    Yao, C. X.; Turecek, F. Hypervalent Ammonium Radicals: Competitive N—C and N—H Bond Dissociations in Methyl Ammonium and Ethyl Ammonium. Phys. Chem. Chem. Phys. 2005, 7, 912–920.CrossRefGoogle Scholar
  16. 16.
    Syrstad, E. A.; Stephens, D. D.; Turecek, F. Hydrogen Atom Adducts to the Amide Bond: Generation and Energetics of Amide Radicals in the Gas Phase. J. Phys. Chem. A. 2003, 107, 115–126.CrossRefGoogle Scholar
  17. 17.
    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
  18. 18.
    Horn, D. M.; Breuker, K.; Frank, A. J.; McLafferty, F. W. Kinetic Intermediates in the Folding of Gaseous Protein Ions Characterized by Electron Capture Dissociation Mass Spectrometry. J. Am. Chem. Soc. 2001, 123, 9792–9799.CrossRefGoogle Scholar
  19. 19.
    Syrstad, E. A.; Vivekananda, S.; Turecek, F. Direct Observation of a Hydrogen Atom Adduct to C-5 in Uracil: A Neutralization-Reionization Mass Spectrometric and ab initio Study. J. Phys. Chem. A. 2001, 105, 8339–8351.CrossRefGoogle Scholar
  20. 20.
    Lin, C.; Cournoyer, J. J.; O’Connor, P. B. Use of a Double Resonance Electron Capture Dissociation Experiment to Probe Fragment Intermediate Lifetimes. J. Am. Soc. Mass Spectrom. 2006, in press.Google Scholar
  21. 21.
    Pashkova, A.; Moskovets, E.; Karger, B. L. Coumarin Tags for Improved Analysis of Peptides by MALDI-TOF MS and MS/MS. 1: Enhancement in MALDI MS Signal Intensities. Anal. Chem. 2004, 76, 4550–4557.CrossRefGoogle Scholar
  22. 22.
    O’Connor, P. B.; Pittman, J. L.; Thomson, B. A.; Budnik, B. A.; Cournoyer, J. C.; Jebanathirajah, J.; Lin, C.; Moyer, S. A New Hybrid Electrospray Fourier Transform Mass Spectrometer: Design and Performance Characteristics. Rapid Commun. Mass Spectrom. 2006, 20, 259–266.CrossRefGoogle Scholar
  23. 23.
    Jebanathirajah, J. A.; Pittman, J. L.; Thomson, B. A.; Budnik, B. A.; Kaur, P.; Rape, M.; Kirschner, M.; Costello, C. E.; O’Connor, P. B. Characterization of a New qQq-FTICR Mass Spectrometer for Post-translational Modification Analysis and Top-Down Tandem Mass Spectrometry of Whole Proteins. J. Am. Soc. Mass Spectrom. 2005, 16, 1985–1999.CrossRefGoogle Scholar
  24. 24.
    Cooper, H. J.; Hudgins, R. R.; Hakansson, K.; Marshall, A. G. Characterization of Amino Acid Side-Chain Losses in Electron Capture Dissociation. J. Am. Soc. Mass Spectrom. 2002, 13, 241–249.CrossRefGoogle Scholar
  25. 25.
    Kjeldsen, F.; Haselmann, K. F.; Budnik, B. A.; Jensen, F.; Zubarev, R. A. Dissociative Capture of Hot (3–13 eV) Electrons by Polypeptide Polycations: An Efficient Process Accompanied by Secondary Fragmentation. Chem. Phys. Lett. 2002, 356, 201–206.Google Scholar
  26. 26.
    Haselmann, K. F.; Budnik, B. A.; Kjeldsen, F.; Polfer, N. C.; Zubarev, R. A. Can the (M·-X) Region in Electron Capture Dissociation Provide Reliable Information on the Amino Acid Composition of Polypeptides?. Eur. J. Mass Spectrom. 2002, 8, 461–469.CrossRefGoogle Scholar
  27. 27.
    Shaffer, S. A.; Sadilek, M.; Turecek, F. Hypervalent ammonium radicals: Effects of Alkyl Groups and Aromatic Substituents. J. Org. Chem. 1996, 61, 5234–5245.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2006

Authors and Affiliations

  • Marina A. Belyayev
    • 1
  • Jason J. Cournoyer
    • 1
  • Cheng Lin
    • 1
  • Peter B. O’Connor
    • 1
  1. 1.Department of ChemistryBoston UniversityBostonUSA

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