Gas-phase conformation-specific photofragmentation of proline-containing peptide ions

  • Tae-Young Kim
  • Stephen J. Valentine
  • David E. Clemmer
  • James P. Reilly


Singly-protonated proline-containing peptides with N-terminal arginine are photodissociated with vacuum ultraviolet (VUV) light in an ESI linear ion trap/orthogonal-TOF (LIT/o-TOF). When proline is the nth residue from the N-terminus, unusual bn + 2 and an + 2 ions are observed. Their formation is explained by homolytic cleavage of the Cα− C bond in conjunction with a rearrangement of electrons and an amide hydrogen. The latter is facilitated by a proline-stabilized gas-phase peptide conformation.


  1. 1.
    Jarrold, M. F. Unfolding, Refolding, and Hydration of Proteins in the Gas Phase. Acc. Chem. Res. 1999, 32, 360–367.CrossRefGoogle Scholar
  2. 2.
    Hoaglund-Hyzer, C. S.; Counterman, A. E.; Clemmer, D. E. Anhydrous Protein Ions. Chem. Rev. 1999, 99, 3037–3079.CrossRefGoogle Scholar
  3. 3.
    Karas, M.; Hillenkamp, F. Laser Desorption Ionization of Proteins with Molecular Masses Exceeding 10,000 Daltons. Anal. Chem. 1988, 60, 2299–2301.CrossRefGoogle Scholar
  4. 4.
    Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Electrospray Ionization for Mass-Spectrometry of Large Biomolecules. Science 1989, 246, 64–71.CrossRefGoogle Scholar
  5. 5.
    Wood, T. D.; Chorush, R. A.; Wampler, F. M.; Little, D. P.; O’Connor, P. B.; McLafferty, F. W. Gas-Phase Folding and Unfolding of Cytochrome-c Cations. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 2451–2454.CrossRefGoogle Scholar
  6. 6.
    McLafferty, F. W.; Guan, Z. Q.; Haupts, U.; Wood, T. D.; Kelleher, N. L. Gaseous Conformational Structures of Cytochrome c. J. Am. Chem. Soc. 1998, 120, 4732–4740.CrossRefGoogle Scholar
  7. 7.
    Valentine, S. J.; Clemmer, D. E. H/D Exchange Levels of Shape-Resolved Cytochrome c Conformers in the Gas Phase. J. Am. Chem. Soc. 1997, 119, 3558–3566.CrossRefGoogle Scholar
  8. 8.
    Valentine, S. J.; Clemmer, D. E. Temperature-Dependent H/D Exchange of Compact and Elongated Cytochrome c Ions in the Gas Phase. J. Am. Soc. Mass Spectrom. 2002, 13, 506–517.CrossRefGoogle Scholar
  9. 9.
    Lifshitz, C. A Review of Gas-Phase H/D Exchange Experiments: The Protonated Arginine Dimer and Bradykinin Nonapeptide Systems. Int. J. Mass Spectrom. 2004, 234, 63–70.CrossRefGoogle Scholar
  10. 10.
    Mazurek, U.; Engeser, M.. Gas-Phase H/D Exchange of the Protonated Serine Octamer Cluster: “Ion Ping Pong” of Populations A and B. Int. J. Mass Spectrom. 2006, 249, 473–476.CrossRefGoogle Scholar
  11. 11.
    Clemmer, D. E.; Jarrold, M. F. Ion Mobility Measurements and Their Applications to Clusters and Biomolecules. J. Mass Spectrom. 1997, 32, 577–592.CrossRefGoogle Scholar
  12. 12.
    Liu, Y. S.; Valentine, S. J.; Counterman, A. E.; Hoaglund, C. S.; Clemmer, D. E. Injected-Ion Mobility Analysis of Biomolecules. Anal. Chem. 1997, 69, A728-A735.CrossRefGoogle Scholar
  13. 13.
    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
  14. 14.
    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.CrossRefGoogle Scholar
  15. 15.
    Burlet, O.; Yang, C. Y.; Gaskell, S. J. Influence of Cysteine to Cysteic Acid Oxidation on the Collision-Activated Decomposition of Protonated Peptides—Evidence for Intraionic Interactions. J. Am. Soc. Mass Spectrom. 1992, 3, 337–344.CrossRefGoogle Scholar
  16. 16.
    Vachet, R. W.; Asam, M. R.; Glish, G. L. Secondary Interactions Affecting the Dissociation Patterns of Arginine-Containing Peptide Ions. J. Am. Chem. Soc. 1996, 118, 6252–6256.CrossRefGoogle Scholar
  17. 17.
    Tsaprailis, G.; Nair, H.; Somogyi, A.; Wysocki, V. H.; Zhong, W. Q.; 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
  18. 18.
    Schnier, P. D.; Price, W. D.; Jockusch, R. A.; Williams, E. R. Blackbody Infrared Radiative Dissociation of Bradykinin and Its Analogues: Energetics, Dynamics, and Evidence for Salt-Bridge Structures in the Gas Phase. J. Am. Chem. Soc. 1996, 118, 7178–7189.CrossRefGoogle Scholar
  19. 19.
    Kjeldsen, F.; Silivra, O. A.; Zubarev, R. A. Zwitterionic States in Gas-Phase Polypeptide Ions Revealed by 157-nm Ultra-Violet Photodissociation. Chem. Eur. J. 2006, 12, 7920–7928.CrossRefGoogle Scholar
  20. 20.
    MacArthur, M. W.; Thornton, J. M. Influence of Proline Residues on Protein Conformation. J. Mol. Biol. 1991, 218, 397–412.CrossRefGoogle Scholar
  21. 21.
    Vanhoof, G.; Goossens, F.; Demeester, L.; Hendriks, D.; Scharpe, S. Proline Motifs in Peptides and Their Biological Processing. FASEB J. 1995, 9, 736–744.Google Scholar
  22. 22.
    Pal, D.; Chakrabarti, P. Cis Peptide Bonds in Proteins: Residues Involved, Their Conformations, Interactions and Locations. J. Mol. Biol. 1999, 294, 271–288.CrossRefGoogle Scholar
  23. 23.
    Huang, Y. Y.; Triscari, J. M.; Pasa-Tolic, L.; Anderson, G. A.; Lipton, M. S.; Smith, R. D.; Wysocki, V. H. Dissociation Behavior of Doubly-Charged Tryptic Peptides: Correlation of Gas-Phase Cleavage Abundance with Ramachandran Plots. J. Am. Chem. Soc. 2004, 126, 3034–3035.CrossRefGoogle Scholar
  24. 24.
    Harrison, A. G.; Young, A. B. Fragmentation Reactions of Deprotonated Peptides Containing Proline. The Proline Effect. J. Mass Spectrom. 2005, 40, 1173–1186.CrossRefGoogle Scholar
  25. 25.
    Hayakawa, S.; Hashimoto, M.; Matsubara, H.; Turecek, F. Dissecting the Proline Effect: Dissociations of Proline Radicals Formed by Electron Transfer to Protonated Pro-Gly and Gly-Pro Dipeptides in the Gas Phase. J. Am. Chem. Soc. 2007, 129, 7936–7949.CrossRefGoogle Scholar
  26. 26.
    Counterman, A. E.; Clemmer, D. E. Cis-Trans Signatures of Proline-Containing Tryptic Peptides in the Gas Phase. Anal. Chem. 2002, 74, 1946–1951.CrossRefGoogle Scholar
  27. 27.
    Schwartz, B. L.; Bursey, M. M. Some Proline Substituent Effects in the Tandem Mass-Spectrum of Protonated Penta-Alanine. Biol. Mass Spectrom. 1992, 21, 92–96.CrossRefGoogle Scholar
  28. 28.
    Loo, J. A.; Edmonds, C. G.; Smith, R. D. Tandem Mass-Spectrometry of Very Large Molecules. 2. Dissociation of Multiply Charged Proline-Containing Proteins from Electrospray Ionization. Anal. Chem. 1993, 65, 425–438.CrossRefGoogle Scholar
  29. 29.
    Vaisar, T.; Urban, J. Probing the Proline Effect in CID of Protonated Peptides. J. Mass Spectrom. 1996, 31, 1185–1187.CrossRefGoogle Scholar
  30. 30.
    Paizs, B.; Suhai, S. Fragmentation Pathways of Protonated Peptides. Mass Spectrom. Rev. 2005, 24, 508–548.CrossRefGoogle Scholar
  31. 31.
    Breci, L. A.; Tabb, D. L.; Yates, J. R.; Wysocki, V. H. Cleavage N-Terminal to Proline: Analysis of a Database of Peptide Tandem Mass Spectra. Anal. Chem. 2003, 75, 1963–1971.CrossRefGoogle Scholar
  32. 32.
    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.CrossRefGoogle Scholar
  33. 33.
    Kim, T.-Y.; Schwartz, J. C.; Reilly, J. P. Development of a Linear Ion Trap/Orthogonal-Time-of-Flight Mass Spectrometer for Time-Dependent Observation of Product Ions by Ultraviolet Photodissociation of Peptide Ions. Anal. Chem. 2009, 81, 8809–8817.CrossRefGoogle Scholar
  34. 34.
    Zhang, L.; Reilly, J. P. Peptide De Novo Sequencing using 157 nm Photodissociation in a Tandem Time-of-Flight Mass Spectrometer. Anal. Chem. 2010, 82, 898–908.CrossRefGoogle Scholar
  35. 35.
    Liu, X. Y.; Valentine, S. J.; Plasencia, M. D.; Trimpin, S.; Naylor, S.; Clemmer, D. E. Mapping the Human Plasma Proteome by SCX-LC-MS-MS. J. Am. Soc. Mass Spectrom. 2007, 18, 1249–1264.CrossRefGoogle Scholar
  36. 36.
    Koeniger, S. L.; Merenbloom, S. I.; Valentine, S. J.; Jarrold, M. F.; Udseth, H. R.; Smith, R. D.; Clemmer, D. E. An IMS-IMS Analogue of MS-MS. Anal. Chem. 2006, 78, 4161–4174.CrossRefGoogle Scholar
  37. 37.
    Mesleh, M. F.; Hunter, J. M.; Shvartsburg, A. A.; Schatz, G. C.; Jarrold, M. F. Structural Information from Ion Mobility Measurements: Effects of the Long-Range Potential. J. Vhys. Chem. 1996, 100, 16082–16086.CrossRefGoogle Scholar
  38. 38.
    Shvartsburg, A. A.; Jarrold, M. F. An Exact Hard-Spheres Scattering Model for the Mobilities of Polyatomic Ions. Chem. Phys. Lett. 1996, 261, 86–91.CrossRefGoogle Scholar
  39. 39.
    Thompson, M. S.; Cui, W.; Reilly, J. P. Fragmentation of Singly Charged Peptide Ions by Photodissociation at λ = 157 nm. Angew. Chem. Int. Ed. 2004, 43, 4791–4794.Google Scholar
  40. 40.
    Cui, W.; Thompson, M. S.; Reilly, J. P. Pathways of Peptide Ion Fragmentation Induced by Vacuum Ultraviolet Light. J. Am. Soc. Mass Spectrom. 2005, 16, 1384–1398.CrossRefGoogle Scholar
  41. 41.
    Zhang, L. Y.; Cui, W.; Thompson, M. S.; Reilly, J. P. Structures of α-Type Ions Formed in the 157 nm Photodissociation of Singly-Charged Peptide Ions. J. Am. Soc. Mass Spectrom. 2006, 17, 1315–1321.CrossRefGoogle Scholar
  42. 42.
    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
  43. 43.
    Gabelica, V.; Schulz, E.; Karas, M. Internal Energy Build-Up in Matrix-Assisted Laser Desorption/Ionization J. Muss Spectrom. 2004, 39, 579–593.CrossRefGoogle Scholar
  44. 44.
    Gabelica, V.; De Pauw, E. Internal Energy and Fragmentation of Ions Produced in Electrospray Sources. Mass Spectrom. Rev. 2005, 24, 566–587.CrossRefGoogle Scholar
  45. 45.
    Spengler, B.; Kirsch, D.; Kaufmann, R. Metastable Decay of Peptides and Proteins in Matrix-Assisted Laser-Desorption Mass-Spectrometry. Rapid Commun. Mass Spectrom. 1991, 5, 198–202.CrossRefGoogle Scholar
  46. 46.
    Spengler, B. Post-Source Decay Analysis in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry of Biomolecules. J. Mass Spectrom. 1997, 32, 1019–1036.CrossRefGoogle Scholar
  47. 47.
    Schimmel, P. R.; Flory, P. J. Conformational Energies and Configurational Statistics of Co-Polypeptides Containing L-Proline. J. Mol. Biol. 1968, 34, 105–120.CrossRefGoogle Scholar
  48. 48.
    Ramek, M.; Kelterer, A. M.; Teppen, B. J.; Schafer, L. Theoretical Structure Investigations of N-Acetyl-L-Proline Amide. J. Mol. Struct. 1995, 352, 59–70.CrossRefGoogle Scholar
  49. 49.
    Zanni, M. T.; Gnanakaran, S.; Stenger, J.; Hochstrasser, R. M. Heterodyned Two-Dimensional Infrared Spectroscopy of Solvent-Dependent Conformations of Acetylproline-NH2. J. Phys. Chem. B 2001, 105, 6520–6535.CrossRefGoogle Scholar
  50. 50.
    Hahn, S.; Lee, H.; Cho, M. Theoretical Calculations of Infrared Absorption, Vibrational Circular Dichroism, and Two-Dimensional Vibrational Spectra of Acetylproline in Liquids Water and Chloroform. J. Chem. Phys. 2004., 121, 1849–1865.CrossRefGoogle Scholar
  51. 51.
    Lee, K. K.; Hahn, S.; Oh, K. I.; Choi, J. S.; Too, C.; Lee, H.; Han, H. Y.; Cho, M. Structure of N-Acetylproline Amide in Liquid Water: Experimentally Measured and Numerically Simulated Infrared and Vibrational Circular Dichroism Spectra, J. Phys. Chem. B 2006, 110, 18834–18843.CrossRefGoogle Scholar
  52. 52.
    Sul, S.; Karaiskaj, D.; Jiang, Y.; Ge, N. H. Conformations of N-Acetyl-L Prolinamide by Two-Dimensional Infrared Spectroscopy. J. Phys. Chem. B 2006, 110, 19891–19905.CrossRefGoogle Scholar
  53. 53.
    O’Hair, R. A. J. The Role of Nucleophile-Electrophile Interactions in the Unimolecular and Bimolecular Gas-Phase Ion Chemistry of Peptides and Related Systems. J. Mass Spectrom. 2000, 35, 1377–1381.CrossRefGoogle Scholar
  54. 54.
    Wysocki, V. H.; Tsaprailis, G.; Smith, L. L.; Breci, L. A. Special Feature: Commentary—Mobile and Localized Protons: A Framework for Understanding Peptide Dissociation. J. Mass Spectrom. 2000, 35, 1399–1406.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2010

Authors and Affiliations

  • Tae-Young Kim
    • 1
  • Stephen J. Valentine
    • 1
  • David E. Clemmer
    • 1
  • James P. Reilly
    • 1
  1. 1.Department of ChemistryIndiana UniversityBloomingtonUSA
  2. 2.Division of Chemistry and Chemical EngineeringCalifornia Institute of TechnologyPasadenaUSA

Personalised recommendations