Dehydration versus deamination of N-terminal glutamine in collision-induced dissociation of protonated peptides

  • Pedatsur Neta
  • Quan -Long Pu
  • Lisa Kilpatrick
  • Xiaoyu Yang
  • Stephen E. Stein


Some of the most prominent “neutral losses” in peptide ion fragmentation are the loss of ammonia and water from N-terminal glutamine. These processes are studied by electrospray ionization mass spectrometry in singly- and doubly-protonated peptide ions undergoing collision-induced dissociation in a triple quadrupole and in an ion trap instrument. For this study, four sets of peptides were synthesized: (1) QLLLPLLLK and similar peptides with K replaced by R, H, or L, and Q replaced by a number of amino acids, (2) QLnK (n=0, 1, 3, 5, 7, 9, 11), (3) QLnR (n=0, 1, 3, 5, 7, 9), and (4) QLn (n=1, 2, 3, 4, 8). The results for QLLLPLLLK and QLLLPLLLR show that the singly protonated ions undergo loss of ammonia and to a smaller extent loss of water, whereas the doubly protonated ions undergo predominant loss of water. The fast fragmentation next to P (forming the y5 ion) occurs to a larger extent than the neutral losses from the singly protonated ions but much less than the water loss from the doubly protonated ions. The results from these and other peptides show that, in general, when N-terminal glutamine peptides have no “mobile protons”, that is, the number of charges on the peptide is no greater than the number of basic amino acids (K, R, H), deamination is the predominant neutral loss fragmentation, but when mobile protons are present the predominant process is the loss of water. Both of these processes are faster than backbone fragmentation at the proline. These results are rationalized on the basis of resonance stabilization of the two types of five-membered ring products that would be formed in the neutral loss processes; the singly protonated ion yields the more stable neutral pyrrolidinone ring whereas the doubly protonated ion yields the protonated aminopyrroline ring (see Schemes). The generality of these trends is confirmed by analyzing an MS/MS spectra library of peptides derived from tryptic digests of yeast. In the absence of mobile protons, glutamine deamination is the most rapid neutral loss process. For peptides with mobile protons, dehydration from glutamine is far more rapid than from any other amino acid. Most strikingly, end terminal glutamine is by far the most labile source of neutral loss in excess-proton peptides, but not highly exceptional when mobile protons are not available. In addition, rates of deamination are faster in lysine versus arginine C-terminus peptides and 20 times faster in positively charged than negatively charged peptides, demonstrating that these formal neutral loss reactions are not “neutral reactions” but depend on charge state and stability.


  1. 1.
    Paizs, B.; Suhai, S. Fragmentation Pathways of Protonated Peptides. Mass Spectrom. Rev. 2005, 24, 508–548.CrossRefGoogle Scholar
  2. 2.
    Huang, Y.; Triscari, J. M.; Tseng, G. C.; Pasa-Tolic, L.; Lipton, M. S.; Smith, R. D.; Wysocki, V. H. Statistical Characterization of the Charge State and Residue Dependence of Low-Energy CID Peptide Dissociation Patterns. Anal. Chem. 2005, 77, 5800–5813;CrossRefGoogle Scholar
  3. 2.(a)
    Dongre, A. R.; Jones, J. I.; 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.
    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
  5. 4.
    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
  6. 5.
    Polce, M. J.; Ren, D.; Wesdemiotis, C. Dissociation of the Peptide Bond in Protonated Peptides. J. Mass Spectrom. 2000, 35, 1391–1398.CrossRefGoogle Scholar
  7. 6.
    Schlosser, A.; Lehmann, W. D. Five-Membered Ring Formation in Unimolecular Reactions of Peptides: A Key Structural Element Controlling Low-Energy Collision-Induced Dissociation of Peptides. J. Mass Spectrom. 2000, 35, 1382–1390.CrossRefGoogle Scholar
  8. 7.
    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
  9. 8.
    Orlowski, M.; Meister, A. Enzymology of Pyrrolidone Carboxylic Acid. In The Enzymes, Vol. IV; Boyer, P. D., Eds.; Academic Press: New York, 1971; pp 123–151.Google Scholar
  10. 9.
    Baldwin, M. A.; Falick, A. M.; Gibson, B. W.; Prusiner, S. B.; Stahl, N.; Burlingame, A. L. Tandem Mass Spectrometry of Peptides with N-Terminal Glutamine: Studies on a Prion Protein Peptide. J. Am. Soc. Mass Spectrom. 1990, 1, 258–264.CrossRefGoogle Scholar
  11. 10.
    Harrison, A. G. Fragmentation Reactions of Protonated Peptides Containing Glutamine or Glutamic Acid. J. Mass Spectrom. 2003, 38, 174–187.CrossRefGoogle Scholar
  12. 11.
    Witt, M.; Grutzmacher, H.-F. Proton-Bound Dimers of Aliphatic Carboxamides: Gas-Phase Basicity and Dissociation Energy. Int. J. Mass Spectrom. Ion Processes 1997, 165, 49–62.CrossRefGoogle Scholar
  13. 12.
    Hunter, E. P. L.; Lias, S. G. Evaluated Gas-Phase Basicities and Proton Affinities of Molecules: An Update. J. Phys. Chem. Ref. Data 1998, 27, 413–656.CrossRefGoogle Scholar
  14. 13.
    Geoghegan, K. F.; Hoth, L. R.; Tan, D. H.; Borzilleri, K. A.; Withka, J. M.; Boyd, J. G. Cyclization of N-Terminal S-Carbamoylmethylcysteine Causing Loss of 17 Da from Peptides and Extra Peaks in Peptide Maps. J. Proteome Res. 2002, 1, 181–187.CrossRefGoogle Scholar
  15. 14.
    Haller, I.; Mirza, U. A.; Chait, B. T. Collision Induced Dissociation of Peptides: Choice of Collision Parameters. J. Am. Soc. Mass Spectrom. 1996, 7, 677–681.CrossRefGoogle Scholar
  16. 15.
    Stein, S.; Kilpatrick, L.; Neta, P.; Roth, J.; Yang, X. ASMS Abstract; June 2006.Google Scholar
  17. 16.
    Peptide Atlas, Scholar
  18. 17.
    Kapp, E. A.; Schutz, F.; Reid, G. E.; Eddes, J. S.; Moritz, R. L.; O’Hair, R. A. J.; Speed, T. P.; Simpson, R. J. Mining a Tandem Mass Spectrometry Database to Determine the Trends and Global Factors Influencing Peptide Fragmentation. Anal. Chem. 2003, 75, 6251–6262.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2007

Authors and Affiliations

  • Pedatsur Neta
    • 1
  • Quan -Long Pu
    • 1
  • Lisa Kilpatrick
    • 1
    • 2
  • Xiaoyu Yang
    • 1
  • Stephen E. Stein
    • 1
  1. 1.Mass Spectrometry Data Center, Physical and Chemical Properties DivisionNational Institute of Standards and TechnologyGaithersburgUSA
  2. 2.Hollins Marine LaboratoryNational Institutes of Standards and TechnologyCharlestonUSA

Personalised recommendations