Evidence for Sequence Scrambling in Collision-Induced Dissociation of y-Type Fragment Ions

  • Mahsan Miladi
  • Brett Harper
  • Touradj SoloukiEmail author
Research Article


Sequence scrambling from y-type fragment ions has not been previously reported. In a study designed to probe structural variations among b-type fragment ions, it was noted that y fragment ions might also yield sequence-scrambled ions. In this study, we examined the possibility and extent of sequence-scrambled fragment ion generation from collision-induced dissociation (CID) of y-type ions from four peptides (all containing basic residues near the C-terminus) including: AAAAHAA-NH2 (where “A” denotes carbon thirteen (13C1) isotope on the alanine carbonyl group), des-acetylated-α-melanocyte (SYSMEHFRWGKPV-NH2), angiotensin II antipeptide (EGVYVHPV), and glu-fibrinopeptide b (EGVNDNEEGFFSAR). We investigated fragmentation patterns of 32 y-type fragment ions, including y fragment ions with different charge states (+1 to +3) and sizes (3 to 12 amino acids). Sequence-scrambled fragment ions were observed from ~50 % (16 out of 32) of the studied y-type ions. However, observed sequence-scrambled ions had low relative intensities from ~0.1 % to a maximum of ~12 %. We present and discuss potential mechanisms for generation of sequence-scrambled fragment ions. To the best of our knowledge, results on y fragment dissociation presented here provide the first experimental evidence for generation of sequence-scrambled fragments from CID of y ions through intermediate cyclic “b-type” ions.

Key words

Collision-induced dissociation (CID) Mass spectrometry (MS) Peptide fragmentation Sequence scrambling y Fragment 



The Authors thank Baylor University for financial support. They thank Dr. Behrooz Zekvat for helpful discussions on CID data. The authors express their gratitude to reviewers of a previous paper [44] for their thoughtful comments and questions that led us to explore y-fragment ion scramblings.

Supplementary material

13361_2013_714_MOESM1_ESM.doc (444 kb)
ESM 1 (DOC 443 kb)


  1. 1.
    McLafferty, F.W.: Collisional activation mass spectra. Philos. Trans. R Soc. A: Math. Phys. Sci. 293, 93–102 (1979)CrossRefGoogle Scholar
  2. 2.
    Little, D.P., Speir, J.P., Senko, M.W., O'Connor, P.B., McLafferty, F.W.: Infrared multiphoton dissociation of large multiply charged ions for biomolecule sequencing. Anal. Chem. 66, 2809–2815 (1994)CrossRefGoogle Scholar
  3. 3.
    Zubarev, R.A., Kelleher, N.L., McLafferty, F.W.: Electron capture dissociation of multiply charged protein cations. A nonergodic process. J. Am. Chem. Soc. 120, 3265–3266 (1998)CrossRefGoogle Scholar
  4. 4.
    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. 101, 9528–9533 (2004)CrossRefGoogle Scholar
  5. 5.
    Roepstorff, P., Fohlman, J.: Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed. Mass Spectrom. 11, 601 (1984)CrossRefGoogle Scholar
  6. 6.
    Baldwin, M.A.: Protein identification by mass spectrometry: issues to be considered. Mol. Cell. Proteom. 3, 1–9 (2004)CrossRefGoogle Scholar
  7. 7.
    Lam, H., Aebersold, R.: Building and searching tandem mass (MS/MS) spectral libraries for peptide identification in proteomics. Methods 54, 424–431 (2011)CrossRefGoogle Scholar
  8. 8.
    Lubec, G., Afjehi-Sadat, L.: Limitations and pitfalls in protein identification by mass spectrometry. Chem. Rev. 107, 3568–3584 (2007)CrossRefGoogle Scholar
  9. 9.
    Nielsen, M.L., Savitski, M.M., Zubarev, R.A.: Improving protein identification using complementary fragmentation techniques in Fourier transform mass spectrometry. Mol. Cell Proteom. 4, 835–845 (2005)CrossRefGoogle Scholar
  10. 10.
    Yague, J., Paradela, A., Ramos, M., Ogueta, S., Marina, A., Barahona, F., Lopez de Castro, J.A., Vazquez, J.: Peptide rearrangement during quadrupole ion trap fragmentation: added complexity to MS/MS spectra. Anal. Chem. 75, 1524–1535 (2003)CrossRefGoogle Scholar
  11. 11.
    Mujezinovic, N., Raidl, G., Hutchins, J.R., Peters, J.M., Mechtler, K., Eisenhaber, F.: Cleaning of raw peptide MS/MS spectra: improved protein identification following deconvolution of multiply charged peaks, isotope clusters, and removal of background noise. Proteomics 6, 5117–5131 (2006)CrossRefGoogle Scholar
  12. 12.
    Chen, Y., Zhang, J., Xing, G., Zhao, Y.: Mascot-derived false positive peptide identifications revealed by manual analysis of tandem mass spectra. J. Proteome Res. 8, 3141–3147 (2009)CrossRefGoogle Scholar
  13. 13.
    Tang, X.J., Boyd, R.K.: An investigation of fragmentation mechanisms of doubly protonated tryptic peptides. Rapid Commun. Mass Spectrom. 6, 651–657 (1992)CrossRefGoogle Scholar
  14. 14.
    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. 65, 2824–2834 (1993)CrossRefGoogle Scholar
  15. 15.
    Tang, X.J., Boyd, R.K.: Rearrangements of doubly charged acylium ions from lysyl and ornithyl peptides. Rapid Commun. Mass Spectrom. 8, 678–686 (1994)CrossRefGoogle Scholar
  16. 16.
    Harrison, A.G., Young, A.B., Bleiholder, C., Suhai, S., Paizs, B.: Scrambling of sequence information in collision-induced dissociation of peptides. J. Am. Chem. Soc. 128, 10364–10365 (2006)CrossRefGoogle Scholar
  17. 17.
    Paizs, B., Suhai, S.: Fragmentation pathways of protonated peptides. Mass Spectrom. Rev. 24, 508–548 (2005)CrossRefGoogle Scholar
  18. 18.
    Sadygov, R.G., Cociorva, D., Yates III, J.R.: Large-scale database searching using tandem mass spectra: looking up the answer in the back of the book. Nat. Methods 1, 195–202 (2004)CrossRefGoogle Scholar
  19. 19.
    Saminathan, I.S., Wang, X.S., Guo, Y., Krakovska, O., Voisin, S., Hopkinson, A.C., Siu, K.W.: The extent and effects of peptide sequence scrambling via formation of macrocyclic b-ions in model proteins. J. Am. Soc. Mass Spectrom. 21, 2085–2094 (2010)CrossRefGoogle Scholar
  20. 20.
    Goloborodko, A.A., Gorshkov, M.V., Good, D.M., Zubarev, R.A.: Sequence scrambling in shotgun proteomics is negligible. J. Am. Soc. Mass Spectrom. 22, 1121–1124 (2011)CrossRefGoogle Scholar
  21. 21.
    Fattahi, A., Solouki, T.: Conformational analysis of metal complexed model peptides and their fragment ions using FT-ICR MS and gas-phase H/D exchange reactions. Proceedings of the 49th ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, IL, May 2001Google Scholar
  22. 22.
    Somogyi, A.: Probing peptide fragment ion structures by combining sustained off-resonance collision-induced dissociation and gas-phase H/D exchange (SORI-HDX) in Fourier transform ion-cyclotron resonance (FT-ICR) instruments. J. Am. Soc. Mass Spectrom. 19, 1771–1775 (2008)CrossRefGoogle Scholar
  23. 23.
    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. 131, 18272–18282 (2009)CrossRefGoogle Scholar
  24. 24.
    Fattahi, A., Zekavat, B., Solouki, T.: H/D exchange kinetics: experimental evidence for formation of different b fragment ion conformers/isomers during the gas-phase peptide sequencing. J. Am. Soc. Mass Spectrom. 21, 358–369 (2010)CrossRefGoogle Scholar
  25. 25.
    Chen, X., Steill, J.D., Oomens, J., Polfer, N.C.: Oxazolone versus macrocycle structures for leu-enkephalin b2–b4: insights from infrared multiple-photon dissociation spectroscopy and gas-phase hydrogen/deuterium exchange. J. Am. Soc. Mass Spectrom. 21, 1313–1321 (2010)CrossRefGoogle Scholar
  26. 26.
    Bythell, B.J., Somogyi, A., Paizs, B.: What is the structure of b2 ions generated from doubly protonated tryptic peptides? J. Am. Soc. Mass Spectrom. 20, 618–624 (2009)CrossRefGoogle Scholar
  27. 27.
    Bythell, B.J., Knapp-Mohammady, M., Paizs, B., Harrison, A.G.: Effect of the His residue on the cyclization of b-ions. J. Am. Soc. Mass Spectrom. 21, 1352–1363 (2010)CrossRefGoogle Scholar
  28. 28.
    Bythell, B.J., Csonka, I.P., Suhai, S., Barofsky, D.F., Paizs, B.: Gas-phase structure and fragmentation pathways of singly protonated peptides with N-terminal arginine. J. Phys. Chem. B 114, 15092–15105 (2010)CrossRefGoogle Scholar
  29. 29.
    Riba-Garcia, I., Giles, K., Bateman, R.H., Gaskella, 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. 19, 609–613 (2008)CrossRefGoogle Scholar
  30. 30.
    Riba-Garcia, I., Giles, K., Bateman, R.H., Gaskell, S.J.: Studies of peptide a- and b-type fragment ions using stable isotope labeling and integrated ion mobility/tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 19, 1781–1787 (2008)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 112, 1286–1293 (2008)CrossRefGoogle Scholar
  32. 32.
    Yoon, S.H., Chamot-Rooke, J., Perkins, B.R., Hildebrand, A.E., Poutsma, J.C., Wysocki, V.H.: IRMPD spectroscopy shows that AGG forms an oxazolone b2 + ion. J. Am. Chem. Soc. 130, 17644–17645 (2008)CrossRefGoogle Scholar
  33. 33.
    Perkins, B.R., Chamot-Rooke, J., Yoon, S.H., Gucinski, A.C., Somogyi, A., Wysocki, V.H.: Evidence of diketopiperazine and oxazolone structures for HA b2 + ion. J. Am. Chem. Soc. 131, 17528–17529 (2009)CrossRefGoogle Scholar
  34. 34.
    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. 131, 11503–11508 (2009)CrossRefGoogle Scholar
  35. 35.
    Solouki, T., Zekavat, B., Miladi, M.: On the existence of structurally different isobaric bn fragment ions. Proceedings of the 58th ASMS Conference on Mass Spectrometry and Allied Topics, Salt Lake City, UT, May 2010Google Scholar
  36. 36.
    Yalcin, T., Khouw, C., Csizmadia, I.G., Peterson, M.R., Harrison, A.G.: Why are b ions stable species in peptide spectra? J. Am. Soc. Mass Spectrom. 6, 1165–1174 (1995)CrossRefGoogle Scholar
  37. 37.
    Yalcin, T., 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. 7, 233–242 (1996)CrossRefGoogle Scholar
  38. 38.
    Paizs, B., Simonyi, M.: Ring inversion barrier of diazepam and derivatives: an ab initio study. Chirality 11(8), 651–658 (1999)CrossRefGoogle Scholar
  39. 39.
    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. 127, 17154–17155 (2005)CrossRefGoogle Scholar
  40. 40.
    Bythell, B.J., Erlekam, U., Paizs, B., Maitre, P.: Infrared spectroscopy of fragments from doubly protonated tryptic peptides. Chem. Phys. Chem. 10, 883–885 (2009)CrossRefGoogle Scholar
  41. 41.
    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. 20, 334–339 (2009)CrossRefGoogle Scholar
  42. 42.
    Tirado, M., Polfer, N.C.: Defying entropy: forming large head-to-tail macrocycles in the gas phase. Angew. Chem. Int. Ed Engl. 124, 1–4 (2012)CrossRefGoogle Scholar
  43. 43.
    Badman, E.R., Myung, S., Clemmer, D.E.: Gas-phase separations of protein and peptide ion fragments generated by collision-induced dissociation in an ion trap. Anal. Chem. 74, 4889–4894 (2002)CrossRefGoogle Scholar
  44. 44.
    Miladi, M., Zekavat, B., Munisamy, S.M., Solouki, T.: A systematic study on the effect of histidine position and fragment ion size on the formation of bn ions. Int. J. Mass Spectrom. 316/318, 164–173 (2012)CrossRefGoogle Scholar
  45. 45.
    Makarov, A., Denisov, E., Kholomeev, A., Balschun, W., Lange, O., Strupat, K., Horning, S.: Performance evaluation of a hybrid linear ion trap/orbitrap mass spectrometer. Anal. Chem. 78(7), 2113–2120 (2006)CrossRefGoogle Scholar
  46. 46.
    Harrison, A.G.: Peptide sequence scrambling through cyclization of b5 ions. J. Am. Soc. Mass Spectrom. 19, 1776–1780 (2008)CrossRefGoogle Scholar
  47. 47.
    Yalcin, T., Harrison, A.G.: Ion chemistry of protonated lysine derivatives. J. Mass Spectrom. 31, 1237–1243 (1996)CrossRefGoogle Scholar
  48. 48.
    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. 75, 1963–1971 (2003)CrossRefGoogle Scholar
  49. 49.
    Kish, M.M., Wesdemiotis, C.: Selective cleavage at internal lysine residues in protonated vs metalated peptides. Int. J. Mass Spectrom. 227, 191–203 (2003)CrossRefGoogle Scholar
  50. 50.
    Tsaprailis, G., Nair, H., Zhong, W., Kuppannan, K., Futrell, J.H., Wysocki, V.H.: A mechanistic investigation of the enhanced cleavage at histidine in the gas-phase dissociation of protonated peptides. Anal. Chem. 76, 2083–2094 (2004)CrossRefGoogle Scholar
  51. 51.
    Bythell, B.J., Maitre, P., Paizs, B.: Cyclization and rearrangement reactions of an fragment ions of protonated peptides. J. Am. Chem. Soc. 132, 14766–14779 (2010)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2013

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistryBaylor UniversityWacoUSA
  2. 2.Institute of Biomedical StudiesBaylor UniversityWacoUSA

Personalised recommendations