Abstract
Photodissociation of iodo-tyrosine modified peptides yields localized radicals on the tyrosine side chain, which can be further dissociated by collisional activation. We have performed extensive experiments on model peptides, RGYALG, RGYG, and their derivatives, to elucidate the mechanisms underlying backbone fragmentation at tyrosine. Neither acetylation nor deuteration of the tyrosyl phenolic hydrogen significantly affects backbone fragmentation. However, deuterium migration from the tyrosyl β carbon is concomitant with cleavage at tyrosine. Substitution of tyrosine with 4-hydroxyphenylglycine, which does not have β hydrogens, results in almost complete elimination of backbone fragmentation at tyrosine. These results suggest that a radical situated on the β carbon is required for a-type fragmentation in hydrogen-deficient radical peptides. Replacement of the αH of the residue adjacent to tyrosine with methyl groups results in significant diminution of backbone fragmentation. The initial radical abstracts an αH from the adjacent amino acid, which is poised to “rebound” and abstract the βH of tyrosine through a six-membered transition-state. Subsequent β-scission leads to the observed a-type backbone fragment. These results from deuterated peptides clearly reveal that radical migration in peptides can occur and that multiple migrations are not infrequent. Counterintuitively, close examination of all experimental results reveals that the probability for fragmentation at a particular residue is well correlated with thermodynamic radical stability. A-type fragmentation therefore appears to be most likely when favorable thermodynamics are combined with the relevant kinetic control. These results are consistent with ab initio calculations, which demonstrate that barriers to migration are significantly smaller in magnitude than probable dissociation thresholds.
Article PDF
Similar content being viewed by others
References
Stubbe, J.; van der Donk, W.A. Protein Radicals in Enzyme Catalysis. Chem. Rev. 1998, 98, 705–762.
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.
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.
Syrstad, E. A.; Turecek, F. Toward a general mechanism of electron capture dissociation. J. Am. Soc. Mass Spectrom. 2005, 16, 208–224.
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.
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.
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.
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.
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.
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.
Prell, J. S.; O’Brien, J. T.; Holm, A. I. S.; Leib, R. D.; Donald, W. A.; Williams, E. R. Electron capture by a hydrated gaseous peptide: effects of water on fragmentation and molecular survival. J. Am. Chem. Soc. 2008, 130, 12680–12689.
Wee, S.; Mortimer, A.; Moran, D.; Wright, A.; Barlow, C. K.; O’Hair, R. A. J.; Radom, L.; Easton, C. J. Gas-phase regio-controlled generation of charged amino acid and peptide radicals. Chem. Commun. 2006, 40, 4233–4235.
Masterson, D. S.; Yin, H. Y.; Chacon, A.; Hachey, D. L.; Norris, J. L.; Porter, N. A. Lysine peroxycarbamates: Free radical-promoted peptide cleavage. J. Am. Chem. Soc. 2004, 126, 720–721.
Hodyss, R.; Cox, H. A.; Beauchamp, J. L. Bioconjugates for Tunable Peptide Sequencing: Free Radical Initiated Peptide Sequencing (FRIPS). J. Am. Chem. Soc. 2005, 127, 12436–12437.
Chu, I. K.; Rodriquez, C. F.; Lau, T. C.; Hopkinson, A. C.; Siu, K. W. M. Molecular Radical Cations of Oligopeptides. J. Phys. Chem. B 2000, 104, 3393–3397.
Wee, S.; O’Hair, R. A. J.; McFadyen, W. D. Comparing the gas phase fragmentation reactions of protonated and radical cations of the tripeptides GXR. Int. J. Mass Spectrom. 2004, 234, 101–122.
Laskin, J.; Yang, Z.; Chu, I. K. Energetics and Dynamics of Electron Transfer and Proton Transfer in Dissociation of MetalIII(salen)-Peptide Complexes in the Gas Phase. J. Am. Chem. Soc. 2008, 130, 3218–3230.
Thompson, M. S.; Cui, W. D.; Reilly, J. P. Fragmentation of singly charged peptide ions by photodissociation at l = 157 nm. Angew. Chem. Int. Edit. 2004, 43, 4791–4794.
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.
Sun, Q.; Nelson, H.; Ly, T.; Stoltz, B. M.; Julian, R. R. Side-chain chemistry mediates backbone fragmentation in hydrogen deficient peptide radicals. J. Proteome Res., in press.
Diedrich, J. K.; Julian, R. R. Site-specific radical directed dissociation of peptides at phosphorylated residues. J. Am. Chem. Soc. 2008, 130, 12212–12213.
Blanksby, S. J.; Ellison, G. B. Bond dissociation energies of organic molecules. Acc. Chem. Res. 2003, 36, 255–263.
Rauk, A.; Yu, D.; Taylor, J.; Shustov, G. V.; Block, D. A.; Armstrong, D. A. Effects of structure on αC–H bond enthalpies of amino acid residues: relevance to H transfers in enzyme mechanisms and in protein oxidation. Biochemistry 1999, 38, 9089–9096.
Kavita, K.; Das, P. K. Photodissociation of C6H5I, C6F5I, and related iodides in the ultraviolet. J. Chem. Phys. 2002, 117, 2038–2044.
Paquet, A. Introduction of 9-fluorenylmethyloxycarbonyl, trichloroethoxycarbonyl, and benzyloxycarbonyl amine protecting groups into O-unprotected hydroxyamino acids using succinimidyl carbonates. Can. J. Chem. 1982, 60, 976–980.
Carpino, L. A.; Han, G. Y. The 9-fluorenylmethyloxycarbonyl amino-protecting group. J. Org. Chem. 1972, 37, 3404–3408.
Chan, W. C.; White, P. D. Eds. Fmoc Solid Phase Peptide Synthesis: A Practical Approach. Oxford University Press: New York, NY, 2004.
Kim, T. Y.; Thompson, M. S.; Reilly, J. P. Peptide photodissociation at 157 nm in a linear ion trap mass spectrometer. Rapid Commun. Mass Spectrom. 2005, 19, 1657–1665.
Gronert, S. Estimation of effective ion temperatures in a quadrupole ion trap. J. Am. Soc. Mass Spectrom. 1998, 9, 845–848.
Peng, C. Y.; Schlegel, H. B. Combining synchronous transit and quasi-Newton methods to find transition states. Israel J. Chem. 1993, 33, 449–454.
Nielsen, M. L.; Budnik, B. A.; Haselmann, K. F.; Olsen, J. V.; Zubarev, R. A. Intramolecular hydrogen atom transfer in hydrogen-deficient polypeptide radical cations. Chem. Phys. Lett. 2000, 330(5/6), 558–562.
Kenny, P. T. M.; Nomoto, K.; Orlando, R. Fragmentation Studies of Peptides — the Formation of Y-Ions. Rapid Commun. Mass Spectrom. 1992, 6(2), 95–97.
Harrison, A. G.; Yalcin, T. Proton mobility in protonated amino acids and peptides. Int. J. Mass Spectrom. Ion Processes 1997, 165, 339–347.
Dunlop, J. R.; Tully, F. P. A kinetic study of OH radical reactions with methane and perdeuterated methane. J. Phys. Chem. 1993, 97, 11148–11150.
Laskin, J.; Futrell, J. H.; Chu, I. K. Is dissociation of peptide radical cations an ergodic process? J. Am. Chem. Soc. 2007, 129, 9598–9599.
Moran, D.; Jacob, R.; Wood, G. P. F.; Coote, M. L.; Davies, M. J.; O’Hair, R. A. J.; Easton, C. J.; Radom, L. Rearrangements in model peptide-type radicals via intramolecular hydrogen-atom transfer. Helv. Chim. Acta 2006, 89, 2254–2272.
Fung, W. M. E.; Chan, T. W. D. Experimental and theoretical investigations of the loss of amino acid side chains in electron capture dissociation of model peptides. J. Am. Soc. Mass Spectrom. 2005, 16, 1523–1535.
Panja, S.; Nielsen, S. B.; Hvelplund, P.; Turecek, F. Inverse hydrogen migration in arginine-containing peptide ions upon electron transfer. J. Am. Soc. Mass Spectrom. 2008, 19, 1726–1742.
Jing, L.; Nash, J. J.; Kenttamaa, H. I. Correlation of hydrogen-atom abstraction reaction efficiencies for aryl radicals with their vertical electron affinities and vertical ionization energies of the hydrogen-atom donors. J. Am. Chem. Soc. 2008, 130, 17697–17709.
Paizs, B.; Suhai, S. Towards understanding the tandem mass spectra of protonated oligopeptides: 1: Mechanism of amide bond cleavage. J. Am. Soc. Mass Spectrom. 2004, 15, 103–113.
Lynch, B. J.; Fast, P. L.; Harris, M.; Truhlar, D. G. Adiabatic connection for kinetics. J. Phys. Chem. A 2000, 104, 4811–4815.
Lingwood, M.; Hammond, J. R.; Hrovat, D. A.; Mayer, J. M.; Borden, J. MPW1K performs much better than B3LYP in DFT calculations on reactions that proceed by proton-coupled electron transfer (PCET). Chem. Theory Comput. 2006, 2, 740–745.
Laskin, J.; Yang, Z.; Lam, C.; Chu, I. K. Charge-remote fragmentation of odd-electron peptide ions. Anal. Chem. 2007, 79, 6607–6614.
Wood, G. P. F.; Easton, C. J.; Rauk, A.; Davies, M. J.; Radom, L. Effect of side chains on competing pathways for β-scission reactions of peptide-backbone alkoxyl radicals. J. Phys. Chem. A 2006, 110, 10316–10323.
Author information
Authors and Affiliations
Corresponding author
Additional information
Published online February 12, 2009
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Ly, T., Julian, R.R. Tracking radical migration in large hydrogen deficient peptides with covalent labels: Facile movement does not equal indiscriminate fragmentation. J Am Soc Mass Spectrom 20, 1148–1158 (2009). https://doi.org/10.1016/j.jasms.2009.02.009
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1016/j.jasms.2009.02.009