Abstract
Electron capture dissociation (ECD) studies of two modified amyloid β peptides (20–29 and 25–35) were performed to investigate the role of H· radicals in the ECD of peptide ions and the free-radical cascade (FRC) mechanism. 2,4,6-Trimethylpyridinium (TMP) was used as the fixed charge tag, which is postulated to both trap the originally formed radical upon electron capture and inhibit the H· generation. It was found that both the number and locations of the fixed charge groups influenced the backbone and side-chain cleavages of these peptides in ECD. In general, the frequency and extent of backbone cleavages decreased and those of side-chain cleavages increased with the addition of fixed charge tags. A singly labeled peptide with the tag group farther away from the protonated site experienced a smaller abundance decrease in backbone cleavage fragments than the one with the tag group closer to the protonated site. Despite the nonprotonated nature of all charge carriers in doubly labeled peptide ions, several c and z· ions were still observed in their ECD spectra. Thus, although H· transfer may be important for the N-Cα bond cleavage, there also exist other pathways, which would require a radical migration via H· abstraction through space or via an amide superbase mechanism. Finally, internal fragment ions were observed in the ECD of these linear peptides, indicating that the important role of the FRC in backbone cleavages is not limited to the ECD of cyclic peptides.
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Little, D. P.; Speir, J. P.; Senko, M. W.; Oconnor, P. B.; McLafferty, F. W. Infrared Multiphoton Dissociation of Large Multiply-Charged Ions for Biomolecule Sequencing. Anal. Chem. 1994, 66, 2809–2815.
Gauthier, J. W.; Trautman, T. R.; Jacobson, D. B. Sustained Off-Resonance Irradiation for Collision-Activated Dissociation Involving Fourier-Transform Mass-Spectrometry-Collision-Activated Dissociation Technique That Emulates Infrared Multiphoton Dissociation. Anal. Chim. Acta 1991, 246, 211–225.
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.
Kelleher, R. L.; Zubarev, R. A.; Bush, K.; Furie, B.; Furie, B. C.; McLafferty, F. W.; Walsh, C. T. Localization of Labile Posttranslational Modifications by Electron Capture Dissociation: The Case of gamma-Carboxyglutamic Acid. Anal. Chem. 1999, 71, 4250–4253.
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.
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.
Cooper, H. J.; Hakansson, K.; Marshall, A. G. The Role of Electron Capture Dissociation in Biomolecular Analysis. Mass Spectrom. Rev. 2005, 24, 201–222.
Liu, H.; Hakansson, K. Electron Capture Dissociation of Tyrosine O-Sulfated Peptides Complexed with Divalent Metal Cations. Anal. Chem. 2006, 78, 7570–7576.
Horn, D. M.; Ge, Y.; McLafferty, F. W. Activated Ion Electron Capture Dissociation for Mass Spectral Sequencing of Larger (42 kDa) Proteins. Anal. Chem. 2000, 72, 4778–4784.
Breuker, K.; Oh, H. B.; 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.
Lin, C.; O’Connor, P. B.; Cournoyer, J. J. Use of a Double Resonance Electron Capture Dissociation Experiment to Probe Fragment Intermediate Lifetimes. J. Am. Soc. Mass Spectrom. 2006, 17, 1605–1615.
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, 14432–14433.
Lin, C.; Cournoyer, J. C.; O’Connor, P. B. Probing the Gas Phase Folding Kinetics of Peptide Ions by IR Activated DR-ECD. J. Am. Soc. Mass Spectrom. 2008, 19, 780–789.
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.
Zubarev, R. A. Reactions of Polypeptide Ions with Electrons in the Gas Phase. Mass Spectrom. Rev. 2003, 22, 57–77.
Bakhtiar, R.; Guan, Z. Q. Electron Capture Dissociation Mass Spectrometry in Characterization of Peptides and Proteins. Biotechnol. Lett. 2006, 28, 1047–1059.
Zubarev, R. A.; Haselmann, K. F.; Budnik, B.; Kjeldsen, F.; Jensen, F. Towards an Understanding of the Mechanism of Electron-Capture Dissociation: A Historical Perspective and Modern Ideas. Eur. J. Mass Spectrom. 2002, 8, 337–349.
Meng, F. Y.; Forbes, A. J.; Miller, L. M.; Kelleher, N. L. Detection and Localization of Protein Modifications by High Resolution Tandem Mass Spectrometry. Mass Spectrom. Rev. 2005, 24, 126–134.
Syrstad, E. A.; Turecek, F. Toward a General Mechanism of Electron Capture Dissociation. J. Am. Soc. Mass Spectrom. 2005, 16, 208–224.
Breuker, K.; Oh, H. B.; Cerda, B. A.; Horn, D. M.; McLafferty, F. W. Hydrogen Atom Loss in Electron-Capture Dissociation: A Fourier Transform-Ion Cyclotron Resonance Study with Single Isotopomeric Ubiquitin Ions. Eur. J. Mass Spectrom. 2002, 8, 177–180.
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.
Cooper, H. J.; Hakansson, K.; Marshall, A. G.; Hudgins, R. R.; Haselmann, K. F.; Kjeldsen, F.; Budnik, B. A.; Polfer, N. C.; Zubarev, R. A. Letter: The Diagnostic Value of Amino Acid Side-Chain Losses in Electron Capture Dissociation of Polypeptides: Comment on: “Can the (M-Center Dot-X) Region in Electron Capture Dissociation Provide Reliable Information on Amino Acid Composition of Polypeptides?”. [Eur. J. Mass Spectrom. 2002, 8, 461–469]. Eur. J. Mass Spectrom. 2003, 9, 221–222.
Savitski, M. M.; Nielsen, M. L.; Zubarev, R. A. Side-Chain Losses in Electron Capture Dissociation to Improve Peptide Identification. Anal. Chem. 2007, 79, 2296–2302.
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.
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.
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.
Demirev, P. A. Generation of Hydrogen Radicals for Reactivity Studies in Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Rapid Commun. Mass Spectrom. 2000, 14, 777–781.
Turecek, F. N-C-alpha Bond Dissociation Energies and Kinetics in Amide and Peptide Radicals: Is the Dissociation a Non-Ergodic Process? J. Am. Chem. Soc. 2003, 125, 5954–5963.
Hudgins, R. R.; Hakansson, K.; Quinn, J. P.; Hendrickson, C. L.; Marshall, A. G. Electron Capture Dissociation of Peptides and Proteins Does Not Require a Hydrogen Atom Mechanism. Proceedings of the 50th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, FL, 2000.
Sobczyk, M.; Anusiewicz, W.; Berdys-Kochanska, J.; Sawicka, A.; Skurski, P.; Simons, J. Coulomb-Assisted Dissociative Electron Attachment: Application to a Model Peptide. J. Phys. Chem. A 2005, 109, 250–258.
Chamot-Rooke, J.; Malosse, C.; Frison, G.; Turecek, F. Electron Capture in Charge-Tagged Peptides: Evidence for the Role of Excited Electronic States. J. Am. Soc. Mass Spectrom. 2007, 18, 2146–2161.
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.
Savitski, M. M.; Kjeldsen, F.; Nielsen, M. L.; Zubarev, R. A. Hydrogen rearrangement to and from radical z fragments in electron capture dissociation of peptides. J. Am. Soc. Mass Spectrom. 2007, 18, 113–120.
Tsybin, O. Y.; He, H.; Emmett, M. R.; Hendrickson, C. L.; Marshall, A. G. Ion Activation in Electron Capture Dissociation Can Distinguish Between N-terminal and C-terminal Product Ions. Anal. Chem. 2007, 79, 7596–7602.
Kjeldsen, F.; Zubarev, R. Secondary Losses via gamma-Lactam Formation in Hot Electron Capture Dissociation: A Missing Link to Complete De Novo Sequencing of proteins? J. Am. Chem. Soc. 2003, 125, 6628–6629.
Tsybin, Y. O.; Witt, M.; Baykut, G.; Kjeldsen, F.; Hakansson, P. Combined Infrared Multiphoton Dissociation and Electron Capture Dissociation with a Hollow Electron Beam in Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Rapid Commun. Mass Spectrom. 2003, 17, 1759–1768.
Belyayev, M. A.; Cournoyer, J. J.; Lin, C.; O’Connor, P. B. The Effect of Radical Trap Moieties on Electron Capture Dissociation Spectra of Substance P. J. Am. Soc. Mass Spectrom. 2006, 17, 1428–1436.
Jones, J. W.; Sasaki, T.; Goodlett, D. R.; Turecek, F. Electron Capture in Spin-Trap Capped Peptides: An Experimental Example of Ergodic Dissociation in Peptide Cation-Radicals. J. Am. Soc. Mass Spectrom. 2007, 18, 432–444.
Roth, K. D. W.; Huang, Z. H.; Sadagopan, N.; Watson, J. T. Charge Derivatization of Peptides for Analysis by Mass Spectrometry. Mass Spectrom. Rev. 1998, 17, 255–274.
Wagner, D. S.; Salari, A.; Gage, D. A.; Leykam, J.; Fetter, J.; Hollingsworth, R.; Watson, J. T. Derivatization of Peptides to Enhance Ionization Efficiency and Control Fragmentation during Analysis by Fast-Atom-Bombardment Tandem Mass-Spectrometry. Biol. Mass Spectrom. 1991, 20, 419–425.
Zaia, J.; Biemann, K. Comparison of Charged Derivatives for High-Energy Collision-Induced Dissociation Tandem Mass-Spectrometry. J. Am. Soc. Mass Spectrom. 1995, 6, 428–436.
Vath, J. E.; Biemann, K. Microderivatization of Peptides by Placing a Fixed Positive Charge at the N-Terminus to Modify High-Energy Collision Fragmentation. Int. J. Mass Spectrom. Ion Process. 1990, 100, 287–299.
Adamczyk, M.; Gebler, J. C.; Wu, J. Charge Derivatization of Peptides to Simplify Their Sequencing with an Ion Trap Mass Spectrometer. Rapid Commun. Mass Spectrom. 1999, 13, 1413–1422.
Chagit, D.; Rabkin, E.; Tsoglin, A. A New Charge Derivatization Procedure for Peptide Sequencing. Org. Biomol. Chem. 2005, 3, 2503–2504.
Syka, J. E. P.; 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. 2004, 101, 9528–9533.
Chamot-Rooke, J.; van der Rest, G.; Dalleu, A.; Bay, S.; Lemoine, J. The Combination of Electron Capture Dissociation and Fixed Charge Derivatization Increases Sequence Coverage for O-Glycosylated and O-Phosphorylated Peptides. J. Am. Soc. Mass Spectrom. 2007, 18, 1405–1413.
Xia, Y.; Gunawardena, H. P.; Erickson, D. E.; McLuckey, S. A. Effects of Cation Charge-Site Identity and Position on Electron-Transfer Dissociation of Polypeptide Cations. J. Am. Chem. Soc. 2007, 129, 12232–12243.
Gunawardena, H. P.; Gorenstein, L.; Erickson, D. E.; Xia, Y.; McLuckey, S. A. Electron Transfer Dissociation of Multiply Protonated and Fixed Charge Disulfide Linked Polypeptides. Int. J. Mass Spectrom. 2007, 265, 130–138.
O’Connor, P. B.; Pittman, J. L.; Thomson, B. A.; Budnik, B. A.; Cournoyer, J. C.; Jebanathirajah, J.; Lin, C.; Moyer, S.; Zhao, C. A New Hybrid Electrospray Fourier Transform Mass Spectrometer: Design and Performance Characteristics. Rapid Commun. Mass Spectrom. 2006, 20, 259–266.
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.
Johnson, R. S. Determination of Peptide and Protein Structure by Tandem Mass Spectrometry (Ph. D. Dissertation). Massachusetts Institute of Technology: Cambridge, MA, 1988.
Johnson, R. S.; Martin, S. A.; Biemann, K. Collision-Induced Fragmentation of (M+H)+ Ions of Peptides: Side-Chain Specific Sequence Ions. Int. J. Mass Spectrom. Ion Process. 1988, 86, 137–154.
King, L. C.; Ozog, F. J. Reactions of Pyrylium and Pyridinium Salts with Amines. J. Org. Chem. 1955, 20, 448–454.
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.
Cooper, H. J. Investigation of the Presence of b Ions in Electron Capture Dissociation Mass Spectra. J. Am. Soc. Mass Spectrom. 2005, 16, 1932–1940.
Nguyen, V. Q.; Turecek, F. Gas-Phase Protonation of Pyridine: A Variable-Time Neutralization-Reionization and Ab Initio Study of Pyridinium Radicals. J. Mass Spectrom. 1997, 32, 55–63.
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.
Hodyss, R.; Cox, H. A.; Beauchamp, J. L. Bioconjugates for Tunable Peptide Fragmentation: Free Radical Initiated Peptide Sequencing (FRIPS). J. Am. Chem. Soc. 2005, 127, 12436–12437.
Ly, T.; Julian, R. R. Residue-Specific Radical-Directed Dissociation of Whole Proteins in the Gas Phase. J. Am. Chem. Soc. 2008, 130, 351–358.
Ly, T.; Julian, R. R. Fast Formation of Sidechain Aryl Radicals by UV Irradiation Produces Selective Fragmentation of Whole Proteins. 2008 Conference on Ion Chemistry and Mass Spectrometry, Lake Arrowhead, CA, 2008.
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Published online June 28, 2008
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Li, X., Cournoyer, J.J., Lin, C. et al. The effect of fixed charge modifications on electron capture dissociation. J Am Soc Mass Spectrom 19, 1514–1526 (2008). https://doi.org/10.1016/j.jasms.2008.06.014
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DOI: https://doi.org/10.1016/j.jasms.2008.06.014