Analytical and Bioanalytical Chemistry

, Volume 392, Issue 5, pp 831–838 | Cite as

Fragmentation of intra-peptide and inter-peptide disulfide bonds of proteolytic peptides by nanoESI collision-induced dissociation

  • Michael Mormann
  • Johannes Eble
  • Christian Schwöppe
  • Rolf M. Mesters
  • Wolfgang E. Berdel
  • Jasna Peter-Katalinić
  • Gottfried Pohlentz
Original Paper


Characterisation and identification of disulfide bridges is an important aspect of structural elucidation of proteins. Covalent cysteine-cysteine contacts within the protein give rise to stabilisation of the native tertiary structure of the molecules. Bottom-up identification and sequencing of proteins by mass spectrometry most frequently involves reductive cleavage and alkylation of disulfide links followed by enzymatic digestion. However, when using this approach, information on cysteine-cysteine contacts within the protein is lost. Mass spectrometric characterisation of peptides containing intra-chain disulfides is a challenging analytical task, because peptide bonds within the disulfide loop are believed to be resistant to fragmentation. In this contribution we show recent results on the fragmentation of intra and inter-peptide disulfide bonds of proteolytic peptides by nano electrospray ionisation collision-induced dissociation (nanoESI CID). Disulfide bridge-containing peptides obtained from proteolytic digests were submitted to low-energy nanoESI CID using a quadrupole time-of-flight (Q-TOF) instrument as a mass analyser. Fragmentation of the gaseous peptide ions gave rise to a set of b and y-type fragment ions which enabled derivation of the sequence of the amino acids located outside the disulfide loop. Surprisingly, careful examination of the fragment-ion spectra of peptide ions comprising an intramolecular disulfide bridge revealed the presence of low-abundance fragment ions formed by the cleavage of peptide bonds within the disulfide loop. These fragmentations are preceded by proton-induced asymmetric cleavage of the disulfide bridge giving rise to a modified cysteine containing a disulfohydryl substituent and a dehydroalanine residue on the C-S cleavage site.


Inter-and intramolecular disulfide bridge Underivatised peptides Low-energy collision-induced dissociation Asymmetric disulfide-bridge cleavage 


  1. 1.
    Creighton TE (1997) Biol Chem 378:731–744CrossRefGoogle Scholar
  2. 2.
    Wedemeyer WJ, Welker E, Narayan M, Scheraga HA (2000) Biochemistry 39:4207–4216CrossRefGoogle Scholar
  3. 3.
    Narayan M, Welker E, Wedemeyer WJ, Scheraga HA (2000) Acc Chem Res 33:805–812CrossRefGoogle Scholar
  4. 4.
    Hogg PJ (2003) Trends Biochem Sci 28:210–214CrossRefGoogle Scholar
  5. 5.
    Brown JR, Hartley BS (1966) Biochemistry 101:214–228Google Scholar
  6. 6.
    Kallestad JC, Shoyab M, Linsley PS (1991) J Biol Chem 266:8940–8945Google Scholar
  7. 7.
    Zhou ZR, Smith DL (1990) Biomed Environ Mass Spectrom 19:782–786CrossRefGoogle Scholar
  8. 8.
    Gorman JJ, Wallis TP, Pitt JJ (2002) Mass Spectrom Rev 21:183–216CrossRefGoogle Scholar
  9. 9.
    Hung CW, Schlosser A, Wei JH, Lehmann WD (2007) Anal Bioanal Chem 389:1003–1016CrossRefGoogle Scholar
  10. 10.
    Köcher T, Engström Å, Zubarev RA (2005) Anal Chem 77:172–177CrossRefGoogle Scholar
  11. 11.
    Qiu XY, Cui M, Lil HL, Liu ZQ, Liu SY (2007) Rapid Commun Mass Spectrom 21:3520–3525CrossRefGoogle Scholar
  12. 12.
    Patterson SD, Katta V (1994) Anal Chem 66:3727–3732CrossRefGoogle Scholar
  13. 13.
    Crimmins DL, Saylor M, Rush J, Thoma RS (1995) Anal Biochem 226:355–361CrossRefGoogle Scholar
  14. 14.
    Gorman JJ, Ferguson BL, Speelman D, Mills J (1997) Protein Sci 6:1308–1315CrossRefGoogle Scholar
  15. 15.
    Zhou J, Ens W, Poppeschriemer N, Standing KG, Westmore JB (1993) Int J Mass Spectrom Ion Proc 126:115–122CrossRefGoogle Scholar
  16. 16.
    Jones MD, Patterson SD, Lu HS (1998) Anal Chem 70:136–143CrossRefGoogle Scholar
  17. 17.
    Schaible V, Wefing S, Resemann A, Suckau D, Bücker A, Wolf-Kümmeth S, Hoffmann D (2002) Anal Chem 74:4980–4988CrossRefGoogle Scholar
  18. 18.
    Zubarev RA, Kruger NA, Fridriksson EK, Lewis MA, Horn DM, Carpenter BK, McLafferty FW (1999) J Am Chem Soc 121:2857–2862CrossRefGoogle Scholar
  19. 19.
    Uggerud E (2004) Int J Mass Spectrom 234:45–50CrossRefGoogle Scholar
  20. 20.
    Mormann M, Maček B, de Peredo AG, Hofsteenge J, Peter-Katalinić J (2004) Int J Mass Spectrom 234:11–21CrossRefGoogle Scholar
  21. 21.
    Adamson JT, Håkansson K (2006) J Proteome Res 5:493–501CrossRefGoogle Scholar
  22. 22.
    Chowdhury SM, Munske GR, Ronald RC, Bruce JE (2007) J Am Soc Mass Spectrom 18:493–501CrossRefGoogle Scholar
  23. 23.
    Kleinnijenhuis AJ, Heck AJR, Duursma MC, Heeren RMA (2005) J Am Soc Mass Spectrom 16:1595–1601CrossRefGoogle Scholar
  24. 24.
    Lioe H, O’Hair RAJ (2007) J Am Soc Mass Spectrom 18:1109–1123CrossRefGoogle Scholar
  25. 25.
    Bean MF, Carr SA (1992) Anal Biochem 201:216–226CrossRefGoogle Scholar
  26. 26.
    Gunawardena HP, O’Hair RAJ, McLuckey SA (2006) J Proteome Res 5:2087–2092CrossRefGoogle Scholar
  27. 27.
    Lioe H, Duan M, O’Hair RAJ (2007) Rapid Commun Mass Spectrom 21:2727–2733CrossRefGoogle Scholar
  28. 28.
    Mihalca R, van der Burgt YEM, Heck AJR, Heeren RMA (2007) J Mass Spectrom 42:450–458CrossRefGoogle Scholar
  29. 29.
    Kim HI, Beauchamp JL (2008) J Am Chem Soc 130:1245–1257CrossRefGoogle Scholar
  30. 30.
    Thakur SS, Balaram P (2007) Rapid Commun Mass Spectrom 21:3420–3426CrossRefGoogle Scholar
  31. 31.
    Papayannopoulos IA (1995) Mass Spectrom Rev 14:49–73CrossRefGoogle Scholar
  32. 32.
    Wang R, Kini RM, Chung MCM (1999) Biochemistry 38:7584–7593CrossRefGoogle Scholar
  33. 33.
    Kessler T, Schwöppe C, Liersch R, Schliemann C, Hintelmann H, Bieker R, Berdel WE, Mesters RM (2008) Curr Drug Disc Technol 5:1–8CrossRefGoogle Scholar
  34. 34.
    Rubino FM, Verduci C, Giampiccolo R, Pulvirenti S, Brambilla G, Colombi A (2004) J Mass Spectrom 39:1408–1416CrossRefGoogle Scholar
  35. 35.
    Fagerquist CK (2004) Rapid Commun Mass Spectrom 18:685–700CrossRefGoogle Scholar
  36. 36.
    Fagerquist CK, Lightfield AR, Lehotay SJ (2005) Anal Chem 77:1473–1482CrossRefGoogle Scholar
  37. 37.
    Esseffar M, Herrero R, Quintanilla E, Dávalos JZ, Jiménez P, Abboud JLM, Yáñez M, Mó O (2007) Chem Eur J 13:1796–1803CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Michael Mormann
    • 1
  • Johannes Eble
    • 2
  • Christian Schwöppe
    • 3
  • Rolf M. Mesters
    • 3
  • Wolfgang E. Berdel
    • 3
  • Jasna Peter-Katalinić
    • 1
  • Gottfried Pohlentz
    • 1
  1. 1.Institute for Medical Physics und BiophysicsUniversity of MünsterMünsterGermany
  2. 2.Institute for Physiological ChemistryUniversity of MünsterMünsterGermany
  3. 3.Department of Medicine/Haematology and OncologyUniversity Hospital of MünsterMünsterGermany

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