Apparent inhibition by arginine of macrocyclic b ion formation from singly charged protonated peptides

  • Samuel P. Molesworth
  • Michael J. Van Stipdonk
Focus: Mobile Proton Model


There is now strong evidence for the existence of macrocyclic isomers of b n + ions, the formation and subsequent opening of which can lead to loss of sequence information from protonated peptides in multiple-stage tandem mass spectrometry experiments. In this study, the fragmentation patterns of protonated YARFLG and permuted isomers of the model peptide were investigated by collision-induced dissociation. Of interest was the potential influence of the arginine residue, and its position in the peptide sequence, on formation of the presumed macrocyclic b5 ion isomer and potential loss of sequence information. We find that regardless of the sequence position (either internal or at the N- or C-terminus), only direct sequence ions or ions directly related to fragmentation of the arginine side chain are observed.


Oxazolone Protonated Peptide Glutamyl Endopeptidase Arginine Side Chain Oxazolone Structure 
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  1. 1.
    Roepstroff, P.; Fohlmann, J. Proposal for a Common Nomenclature for Sequence Ions in Mass Spectra of Peptides. J. Biomed. Mass Spectrom. 1984, 11, 601.CrossRefGoogle Scholar
  2. 2.
    Papayannopoulos, I. A. The Interpretation of Collision-Induced Dissociation Tandem Mass Spectra of Peptides. 1995, 14, 49–73.Google Scholar
  3. 3. (a)
    Dongre, A. R.; Jones, J. L.; Somogyi, A.; 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
  4. 3. (b)
    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
  5. 3. (c)
    Jones, J. L.; Dongré, A. R.; Somogyi, A.; Wysocki, V. H. Sequence Dependence of Peptide Fragmentation Efficiency Curves Determined by Electrospray Ionization/Surface-Induced Dissociation Mass Spectrometry. J. Am. Chem. Soc. 1994, 116, 8368–8369.CrossRefGoogle Scholar
  6. 4. (a)
    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
  7. 4. (b)
    Cox, K. A.; Gaskell, S. J.; Morris, M. Whiting, A. Role of the Site of Protonation in the Low-Energy Decompositions of Gas-Phase Peptide Ions. J. Am. Soc. Mass Spectrom. 1996, 7, 522–531.CrossRefGoogle Scholar
  8. 4. (c)
    Summerfield, S. G.; Whiting, A.; Gaskell, S. J. Intraionic Interactions in Electrosprayed Peptide Ions. Int. J. Mass Spectrom. Ion Processes 1997, 162, 149–161.CrossRefGoogle Scholar
  9. 5.
    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
  10. 6.
    Paizs, B.; Lendvay, G.; Vekey, K.; Suhai, S. Formation of b2+ Ions from Protonated Peptides: An Ab Initio Study. Rapid Commun. Mass Spectrom. 1999, 13, 525–533.CrossRefGoogle Scholar
  11. 7.
    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
  12. 8.
    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
  13. 9.
    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
  14. 10.
    Paizs, B.; Suhai, S. Fragmentation Pathways of Protonated Peptides. Mass Spectrom. Rev. 2004, 24, 508–548.CrossRefGoogle Scholar
  15. 11.
    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
  16. 12.
    Oomens, J.; Young, S.; Molesworth, S.; van Stipdonk, M. Spectroscopic Evidence for an Oxazolone Structure of the b2 Fragment Ion from Protonated Tri-Alanine. J. Am. Soc. Mass Spectrom. 2009, 20, 334–339.CrossRefGoogle Scholar
  17. 13.
    Yoon, S. H.; Chamot-Rooke, J.; Perkins, B. R.; Hilderbrand, A. E.; Poutsma, J. C.; Wysocki, V. H. IRMPD Spectroscopy Shows That AGG Forms an Oxazolone b2+ Ion. J. Am. Chem. Soc. 2008, 130, 17644–17645.CrossRefGoogle Scholar
  18. 14.
    Bythell, B. J.; Erlekam, U.; Paizs, B.; Maître, P. Infrared Spectroscopy of Fragments from Doubly Protonated Tryptic Peptides. Chem. Phys. Chem. 2009, 10, 883–885.Google Scholar
  19. 15.
    Chen, X.; Yu, L.; Steill, J. D.; Oomens, J.; Polfer, N. C. Effect of Peptide Fragment Size on the Propensity of Cyclization in Collision-Induced Dissociation: Oligoglycine b2-b8 J. Am. Chem. Soc. 2009, 131, 18272–18282.CrossRefGoogle Scholar
  20. 16.
    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, 7154–17155.Google Scholar
  21. 17.
    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
  22. 18.
    Riba-Garcia, F.; 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. 19.
    Bleiholder, C.; Osburn, S.; Williams, T. D.; Suhai, S.; Van Stipdonk, M.; Flarrison, A. G.; Paizs, B. Sequence Scrambling Fragmentation Pathways of Protonated Peptides. J. Am. Chem. Soc. 2008, 130, 17774–17789.CrossRefGoogle Scholar
  24. 20.
    Jia, C.; Qi, W.; He, Z. Cyclization Reaction of Peptide Fragment Ions during Multistage Collisionally Activated Decomposition: An Inducement to Lose Internal Amino-Acid Residues. J. Am. Soc. Mass Spectrom. 2007, 18, 663–667.CrossRefGoogle Scholar
  25. 21.
    Molesworth, S.; Osburn, S.; Van Stipdonk, M. Influence of Size on Apparent Scrambling of Sequence During CID of b-Type Ions. J. Am. Soc. Mass Spectrom. 2009, 20, 2174–2181.CrossRefGoogle Scholar
  26. 22.
    Harrison, A. Cyclization of Peptide b9 Ions. J. Am. Soc. Mass Spectrom. 2009, 20, 2248–2253.CrossRefGoogle Scholar
  27. 23.
    Erlekam, U.; Bythell, B. J.; Scuderi, D.; Van Stipdonk, M.; Paizs, B.; Maitre, P. Infrared Spectroscopy of Fragments of Protonated Peptides: Direct Evidence for Macrocyclic Structures of b5 Ions. J. Am. Chem. Soc. 2009, 131, 11503–11508.CrossRefGoogle Scholar
  28. 24.
    Olsen, J. V.; Mann, M. Improved Peptide Identification in Proteomics by Two Consecutive Stages of Mass Spectrometric Fragmentation. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 13417–13422.CrossRefGoogle Scholar
  29. 25.
    Farrugia, J. M.; O’Hair, R. A. J. Involvement of Salt Bridges in a Novel Gas Phase Rearrangement of Protonated Arginine-Containing Dipeptides, Which Precedes Fragmentation. Int. J. Mass Spectrom. 2003, 222, 229–242.CrossRefGoogle Scholar
  30. 26.
    Paizs, B.; Suhai, S.; Hargittai, B.; Hruby, V. J.; Somogyi, A. Ab Initio and MS/MS Studies on Protonated Peptides Containing Basic and Acidic Amino Acid Residues: I. Solvated Proton vs. Salt-Bridged Structures and the Cleavage of the Terminal Amide Bond of Protonated RD-NH2. Int. J. Mass Spectrom. 2002, 219, 203–232.CrossRefGoogle Scholar
  31. 27.
    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 Methylester Model Systems. Int. J. Mass Spectrom. 2001, 210/211, 71–87.CrossRefGoogle Scholar
  32. 28.
    Yalcin, T.; Harrison, A. G. Ion Chemistry of Protonated Lysine Derivatives. J. Mass Spectrom. 1996, 31, 1237–1243.CrossRefGoogle Scholar
  33. 29.
    Kish, M. M.; Wesdemiotis, C. Selective Cleavage at Internal Lysine Residues in Protonated vs. Metalated Peptides. Int. J. Mass Spectrom. 2003, 227, 191–203.CrossRefGoogle Scholar
  34. 30.
    Molesworth, S.; Leavitt, C. M.; Groenewold, G. S.; Oomens, J.; Steill, J.; Van Stipdonk, M. Spectroscopic Evidence for Mobilization of Amide Position Protons During CID of Model Peptide Ions. J. Am. Soc. Mass Spectrom. 2009, 20, 1841–1845.CrossRefGoogle Scholar
  35. 31.
    Bythell, B. J.; Suhai, S.; Somogyi, A.; Paizs, B. Proton-Driven Amide Bond-Cleavage Pathways of Gas-Phase Peptide Ions Lacking Mobile Protons. J. Am. Chem. Soc. 2009, 131, 14057–14065.CrossRefGoogle Scholar
  36. 32.
    Molesworth, S.; Osburn, S.; Van Stipdonk, M. Influence of Amino Acid Side Chains on Apparent Selective Opening of Cyclic b5 Ions. J. Am. Soc. Mass Spectrom. 2009, unpublished, (submitted).Google Scholar
  37. 33.
    Dormady, S. J.; Lei, J.; Regnier, F. E. Eliminating Disulfide Exchange During Glutamyl Endopeptidase Digestion of Native Protein. J. Chromatogr. A 1999, 864, 237–245.CrossRefGoogle Scholar
  38. 34.
    Milgotina, E. I.; Voyushina, T. L.; Chestukhina, G. G. Glutamyl Endopeptidases: Structure, Function, and Practical Application. Russ. J. Bioorgan. Chem. 2003, 29, 511–522.CrossRefGoogle Scholar
  39. 35.
    Houmard, J.; Drapeau, G. R. Staphylococcal Protease: A Proteolytic Enzyme Specific for Glutamoyl Bonds. Proc. Nat. Acad. Sci. U.S.A. 1972, 69, 3506–3509.CrossRefGoogle Scholar
  40. 36.
    Fernandez, J.; Mische, S. M. Enzymatic Digestion of Membrane-Bound Proteins for Peptide Mapping and Internal Sequence Analysis. In The. Protein Protocols Handbook 2nd ed., Walker, J. M. Ed.; Humana Press: Totowa, NJ, p. 523–532.Google Scholar
  41. 37.
    Chan, W. C.; White, P. D. Fmoc Solid Phase Peptide Synthesis—A Practical Approach; Oxford University Press: New York, 2000.Google Scholar
  42. 38.
    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

Copyright information

© American Society for Mass Spectrometry 2010

Authors and Affiliations

  • Samuel P. Molesworth
    • 1
  • Michael J. Van Stipdonk
    • 1
  1. 1.Department of ChemistryWichita State UniversityWichitaUSA

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