Journal of the American Society for Mass Spectrometry

, Volume 19, Issue 12, pp 1788–1798

Structure and reactivity of an and a*n peptide fragments investigated using isotope labeling, tandem mass spectrometry, and density functional theory calculations

  • Benjamin J. Bythell
  • Samuel Molesworth
  • Sandra Osburn
  • Travis Cooper
  • Béla Paizs
  • Michael Van Stipdonk
Focus: Peptide Fragmentation

Abstract

Extensive 15N labeling and multiple-stage tandem mass spectrometry were used to investigate the fragmentation pathways of the model peptide FGGFL during low-energy collision-induced-dissociation (CID) in an ion-trap mass spectrometer. Of particular interest was formation of a4 from b4 and a*4 (a4-NH3) from a4 ions correspondingly, and apparent rearrangement and scrambling of peptide sequence during CID. It is suggested that the original FGGFoxab4 structure undergoes b-type scrambling to form GGFFoxa. These two isomers fragment further by elimination of CO and 14NH3 or 15NH3 to form the corresponding a4and a*4 isomers, respectively. For (15N-F)GGFL and FGG(15N-F)L the a*4 ion population appears as two distinct peaks separated by 1 mass unit. These two peaks could be separated and fragmented individually in subsequent CID stages to provide a useful tool for exploration of potential mechanisms along the a4a*4 pathway reported previously in the literature (Vachet et al. J. Am. Chem. Soc.1997, 119, 5481, and Cooper et al. J. Am. Soc. Mass Spectrom.2006, 17, 1654). These mechanisms result in formally the same a*4 structures but differ in the position of the expelled nitrogen atom. Detailed analysis of the observed fragmentation patterns for the separated light and heavy a*4 ion fractions of (15N-F)GGFL indicates that the mechanism proposed by Cooper et al. is consistent with the experimental findings, while the mechanism proposed by Vachet et al. cannot account for the labeling data. In addition, a new rearrangement pathway is presented for a4*-CO ions that effectively transfers the former C-terminal amino acid residue to the N-terminus.

References

  1. 1.
    Hunt, D. F.; Yates, J. R. III; Shabanonowitz, J.; Winston, S.; Hauer, C. R. Protein Sequencing by Tandem Mass Spectrometry. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 6233–6237.CrossRefGoogle Scholar
  2. 2.
    Biemann, K. Contributions of Mass Spectrometry to Peptide and Protein Structure. Biomed. Environ. Mass Spectrom. 1988, 16, 99–111.CrossRefGoogle Scholar
  3. 3.
    Paizs, B.; Suhai, S. Fragmentation Pathways of Protonated Peptides. Mass Spectrom. Rev. 2004, 24, 508–548.CrossRefGoogle Scholar
  4. 4.
    Steen, H.; Mann, M. The abc’s and the xyz’s of Peptide Sequencing. Nat. Rev. Mol. Cell. Biol. 2004, 5, 699–711.CrossRefGoogle Scholar
  5. 5.
    Nesvizhskii, A. E.; Vitek, O.; Aebersold, R. Analysis and Validation of Proteomic Data Generated by Tandem Mass Spectrometry. Nat. Methods 2007, 4, 787–797.CrossRefGoogle Scholar
  6. 6.
    Roepstorff, P.; Fohlmann, J. Proposal for a Common Nomenclature for Sequence Ions in Mass Spectra of Peptides. J. Biomed. Mass Spectrom. 1984, 11601.Google Scholar
  7. 7.
    Biemann, K. Contributions of Mass Spectrometry to Peptide and Protein Structure. Biomed. Environ. Mass Spectrom. 1988, 16, 99.CrossRefGoogle Scholar
  8. 8.
    Dongré, A. R.; Jones, J. L.; Somogyi, Á.; Wysocki, V. H. Influence of Peptide Composition, Gas-Phase Basicity and Chemical Modification on Fragmentation Efficiency: Evidence for the Mobile Proton Model. J. Am. Chem. Soc. 1996, 118, 8365–8374.CrossRefGoogle Scholar
  9. 9.
    Wysocki, V. H.; Tsaprailis, G.; Smith, L. L.; Breci, L. A. Mobile and Localized Protons: A Framework for Understanding Peptide Dissociation. J. Mass Spectrom. 2000, 35, 1399–1406.CrossRefGoogle Scholar
  10. 10.
    Yalcin, T.; Csizmadia, I. G.; Peterson, M. B.; Harrison, A. Why are b-Ions Stable Species in Peptide Spectra? J. Am. Soc. Mass Spectrom. 1996, 7, 233–242.CrossRefGoogle Scholar
  11. 11.
    Paizs, B.; Lendvay, G.; Vékey, K.; Suhai, S. Formation of b2+ Ions from Protonated Peptides: An ab Initio Study. Rapid Commun. Mass Spectrom. 1999, 13, 525–533.CrossRefGoogle Scholar
  12. 12.
    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.CrossRefGoogle Scholar
  13. 13.
    Paizs, B.; Suhai, S. Combined Quantum Chemical and RRKM Modeling of the Main Fragmentation Pathways of Protonated GGG: II. Formation of b2, y1, and y2 ions. Rapid Commun. Mass Spectrom. 2002, 16, 375–389.CrossRefGoogle Scholar
  14. 14.
    Polce, M. J.; Ren, D.; Wesdemiotis, C. Dissociation of the Peptide Bond in Protonated Peptides. J. Mass Spectrom. 2000, 35(12), 1391–1398.CrossRefGoogle Scholar
  15. 15.
    Cordero, M. M.; Houser, J. J.; Wesdemiotis, C. The Neutral Products Formed During Backbone Fragmentations of Protonated Peptides in Tandem Mass Spectrometry. Anal. Chem. 1993, 65, 1594–1601.CrossRefGoogle Scholar
  16. 16.
    Nold, M. J.; Wesdemiotis, C.; Yalcin, T.; Harrison, A. G. Amide Bond Dissociation in protonated Peptides: Structures of the N-terminal Ionic and Neutral Fragments. Int. J. Mass Spectrom. Ion Processes 1997, 164, 137–153.CrossRefGoogle Scholar
  17. 17.
    Yalcin, T.; Khouw, C.; Csizmadia, I. G.; Peterson, M. R.; Harrison, A.G. The Structure and Fragmentation of bn (n ⩾ 3) Ions in Peptide Spectra. J. Am. Soc. Mass Spectrom. 1995, 6, 1165–1174.CrossRefGoogle Scholar
  18. 18.
    Polfer, N. C.; Oomens, J.; Suhai, S.; Paizs, B. Spectroscopic and Theoretical Evidence for Oxazolone Ring Formation in Collision Induced Dissociation of Peptides. J. Am. Chem. Soc. 2005, 127, 17154–17155.CrossRefGoogle Scholar
  19. 19.
    Polfer, N. C.; Oomens, J.; Suhai, S.; Paizs, B. Infrared Spectroscopy and Theoretical Studies on Gas-Phase Protonated Leu-enkephalin and Its Fragments: Direct Experimental Evidence for the Mobile Proton. J. Am. Chem. Soc. 2007, 129, 5887–5897.CrossRefGoogle Scholar
  20. 20.
    Harrison, A. G.; Young, A. B.; Bleiholder, B.; Suhai, S.; Paizs, B. Scrambling of Sequence Information in Collision-Induced Dissociation of Peptides. J. Am. Chem. Soc. 2006, 128, 10364–10365.CrossRefGoogle Scholar
  21. 21.
    Tang, X. -J.; Thibault, P.; Boyd, R. K. Fragmentation Reactions of Multiply-protonated Peptides and Implications for Sequencing by Tandem Mass-spectrometry with Low-Energy Collision-induced Dissociation. Anal. Chem. 1993, 65, 2824–2834.CrossRefGoogle Scholar
  22. 22.
    Riba-Garcia, I.; Giles, K.; Bateman, R. H.; Gaskell, S. J. Evidence for Structural Variants of a- and b-Type Peptide Fragment Ions Using Combined Ion Mobility/Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2008, 19, 609–613.CrossRefGoogle Scholar
  23. 23.
    Farrugia, J. M.; O’Hair, R. A. J.; Reid, G. E. Do All b2 Ions Have Oxazolone Structures?: Multistage Mass Spectrometry and Ab Initio Studies on Protonated N-Acyl Amino Acid Methyl Ester Model Systems. Int. J. Mass Spectrom. Ion Processes 2001, 210/211, 71–87.Google Scholar
  24. 24.
    Gu, C.; Tsaprailis, G.; Breci, L.; Wysocki, V. H. Selective Gas-Phase Cleavage at the Peptide Bond C-Terminal to Aspartic Acid in Fixed-Charge Derivatives of Asp-Containing Peptides. Anal. Chem. 2000, 72, 5804–5813.CrossRefGoogle Scholar
  25. 25.
    Tsaprailis, G.; Nair, H.; Somogyi, A.; Wysocki, V. H.; Zhong, W.; Futrell, J. H.; Summerfield, S. G.; Gaskell, S. J. Influence of Secondary Structure on the Fragmentation of Protonated Peptides. J. Am. Chem. Soc. 1999, 121, 5142–5154.CrossRefGoogle Scholar
  26. 26.
    Farrugia, J. M.; Taverner, T.; O’Hair, R. A. J. Side-Chain Involvement in the Fragmentation Reactions of the Protonated Methyl Esters of Histidine and Its Peptides. 2001, 209, 99–112.Google Scholar
  27. 27.
    Savitski, M. M.; Falth, M.; Eva Fung, Y. M.; Adams, C. M.; Zubarev, R. A Bifurcating Fragmentation Behavior of Gas-Phase Tryptic Peptide Dications in Collisional Activation. J. Am. Soc. Mass Spectrom. (2008), doi. 10.1016/j.jasms.2008.08.003.Google Scholar
  28. 28.
    Vachet, R. W.; Ray, K. L.; Glish, G. L. Origin of Product Ions in the MS/MS Spectra of Peptides in a Quadrupole Ion Trap. J. Am. Soc. Mass Spectrom. 1998, 9, 341–344.CrossRefGoogle Scholar
  29. 29.
    Paizs, B.; Szlavik, Z.; Lendvay, G.; Vékey, K.; Suhai, S. Formation of a2+ ions of protonated peptides: An Ab Initio Study. Rapid Commun. Mass Spectrom. 2000, 14, 746–755.CrossRefGoogle Scholar
  30. 30.
    Vachet, R. W.; Bishop, B. M.; Erickson, B. W.; Glish, G. L. Novel Peptide Dissociation: Gas-Phase Intramolecular Rearrangement of Internal Amino Acid Residues. J. Am. Chem. Soc. 1997, 119, 5481–5488.CrossRefGoogle Scholar
  31. 31.
    Polfer, N. C.; Bohrer, B. C.; Plasencia, M. D.; Paizs, B.; Clemmer, D. E. On the Dynamics of Fragment Isomerization in Collision-Induced Dissociation of Peptides. J. Phys. Chem. A 2008, 112, 1286–1293.CrossRefGoogle Scholar
  32. 32.
    Bleiholder, C.; Osburn, S.; Williams, T. D.; Suhai, S.; Van Stipdonk, M.; Harrison, A. G.; Paizs, B. Sequence Scrambling Fragmentation Pathways of Protonated Peptides, unpublished.Google Scholar
  33. 33.
    Fmoc Solid Phase Peptide Synthesis—A Practical Approach; Chan, W. C.; White, P. D.; Eds; Oxford University Press: New York, 2000.Google Scholar
  34. 34.
    Barr, J. M.; Van Stipdonk, M. J. Multi-Stage Tandem Mass Spectrometry of Metal Cationized Leucine Enkephalin and Leucine Enkephalin Amide. Rapid Commun. Mass Spectrom. 2002, 16, 566–578.CrossRefGoogle Scholar
  35. 35.
    Paizs, B.; Suhai, S. Combined Quantum Chemical and RRKM Modeling of the Main Fragmentation Pathways of Protonated GGG: II. Formation of b2, y1, and y2 ions. Rapid Commun. Mass Spectrom. 2002, 16, 375–389.CrossRefGoogle Scholar
  36. 36.
    Case, D. A.; Pearlman, D. A.; Caldwell, J. W.; Cheatham, T. E., III; Ross, W. S.; Simmerling, C. L.; Darden, T. A.; Merz, K. M.; Stanton, R. V.; Cheng, A. L.; Vincent, J. J.; Crowley, M.; Tsui, V.; Radmer, R. J.; Duan, Y.; Pitera, J.; Massova, I. G.; Seibel, G. L.; Singh, U. C.; Weiner, P. K.; Kollmann, P. A. AMBER 99, University of California: San Francisco, 1999.Google Scholar
  37. 37.
    Gaussian 03, Revision C.02, Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A. III; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; and Pople, J. A.; Gaussian, Inc., Wallingford CT, 2004.Google Scholar
  38. 38.
    Harrison, A. G.; Young, A. B. Fragmentation of Protonated Oligoalanines: Amide Bond Cleavage and Beyond. J. Am. Soc. Mass Spectrom. 2004, 15, 1810–1819.CrossRefGoogle Scholar
  39. 39.
    Cooper, T.; Talaty, E.; Grove, J.; Suhai, S.; Paizs, B.; Van Stipdonk, M. Isotope Labeling and Theoretical Study of the Formation of a*3 Ions from Protonated Tetraglycine. J. Am. Soc. Mass Spectrom. 2006, 17, 1654–1664.CrossRefGoogle Scholar
  40. 40.
    Bythell, B. J.; Barofsky, D. F.; Pingitore, F.; Wang, P.; Wesdemiotis, C.; Paizs, B. Backbone Cleavages and Sequential Loss of Carbon Monoxide and Ammonia from Protonated AGG: A Combined Tandem Mass Spectrometry, Isotope Labeling, and Theoretical Study. J. Am. Soc. Mass Spectrom. 2007, 18, 1291–1303.CrossRefGoogle Scholar
  41. 41.
    Kinser, R. D.; Ridge, D. P.; Hvistendahl, G.; Rasmussen, B.; Uggerud, E. The Unimolecular Chemistry of Protonated Glycinamide and the Proton Affinity of Glycinamide Mass Spectrometric Experiments and Theoretical Model. Chem. Eur. J. 1996, 2, 1143–1149.CrossRefGoogle Scholar
  42. 42.
    Allen, J. M.; Black, D. M.; Johnson, J. S.; Glish, G. L.; Bythell, B. J.; Paizs, B. Why Do b3 Ions not Form a3 Ions? Unpublished.Google Scholar

Copyright information

© American Society for Mass Spectrometry 2008

Authors and Affiliations

  • Benjamin J. Bythell
    • 2
  • Samuel Molesworth
    • 1
  • Sandra Osburn
    • 1
  • Travis Cooper
    • 1
  • Béla Paizs
    • 2
  • Michael Van Stipdonk
    • 1
  1. 1.Department of ChemistryWichita State UniversityWichitaUSA
  2. 2.Department of Molecular BiophysicsGerman Cancer Research CenterHeidelbergGermany

Personalised recommendations