Isotope-labeled cross-linkers and fourier transform ion cyclotron resonance mass spectrometry for structural analysis of a protein/peptide complex

  • Christian Ihling
  • Andreas Schmidt
  • Stefan Kalkhof
  • Daniela M. Schulz
  • Christoph Stingl
  • Karl Mechtler
  • Michael Haack
  • Annette G. Beck-Sickinger
  • Dermot M. F. Cooper
  • Andrea Sinz


For structural studies of proteins and their complexes, chemical cross-linking combined with mass spectrometry presents a promising strategy to obtain structural data of protein interfaces from low quantities of proteins within a short time. We explore the use of isotope-labeled cross-linkers in combination with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry for a more efficient identification of cross-linker containing species. For our studies, we chose the calcium-independent complex between calmodulin and a 25-amino acid peptide from the C-terminal region of adenylyl cyclase 8 containing an “IQ-like motif.” Cross-linking reactions between calmodulin and the peptide were performed in the absence of calcium using the amine-reactive, isotope-labeled (d0 and d4) cross-linkers BS3 (bis[sulfosuccinimidyl]suberate) and BS2G (bis[sulfosuccinimidyl]glutarate). Tryptic in-gel digestion of excised gel bands from covalently cross-linked complexes resulted in complicated peptide mixtures, which were analyzed by nano-HPLC/nano-ESI-FTICR mass spectrometry. In cases where more than one reactive functional group, e.g., amine groups of lysine residues, is present in a sequence stretch, MS/MS analysis is a prerequisite for unambiguously identifying the modified residues. MS/MS experiments revealed two lysine residues in the central α-helix of calmodulin as well as three lysine residues both in the C-terminal and N-terminal lobes of calmodulin to be cross-linked with one single lysine residue of the adenylyl cyclase 8 peptide. Further cross-linking studies will have to be conducted to propose a structural model for the calmodulin/peptide complex, which is formed in the absence of calcium. The combination of using isotope-labeled cross-linkers, determining the accurate mass of intact cross-linked products, and verifying the amino acid sequences of cross-linked species by MS/MS presents a convenient approach that offers the perspective to obtain structural data of protein assemblies within a few days.


  1. 1.
    Sinz, A. Chemical Cross-Linking and Mass Spectrometry for Mapping Three-Dimensional Structures of Proteins and Protein Complexes. J. Mass Spectrom. 2003, 38, 1225–1237.CrossRefGoogle Scholar
  2. 2.
    Back, J. W.; de Jong, L.; Muijsers, A. O.; de Koster, C. G. Chemical Cross-Linking and Mass Spectrometry for Protein Structural Modeling. J. Mol. Biol. 2003, 331, 303–313.CrossRefGoogle Scholar
  3. 3.
    Sinz, A. Chemical Cross-Linking and FTICR Mass Spectrometry for Protein Structure Characterization. Anal. Bioanal. Chem. 2005, 381, 44–47.CrossRefGoogle Scholar
  4. 4.
    Karas, M.; Hillenkamp, F. Laser Desorption Ionization of Proteins with Molecular Masses Exceeding 10,000 Da. Anal. Chem. 1988, 60, 2299–2301.CrossRefGoogle Scholar
  5. 5.
    Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Electrospray Ionization for Mass Spectrometry of Large Biomolecules. Science. 1989, 46, 64–71.CrossRefGoogle Scholar
  6. 6.
    Müller, D. R.; Schindler, P.; Towbin, H.; Wirth, U.; Voshol, H.; Hoving, S.; Steinmetz, M. O. Isotope-Tagged Cross-Linking Reagents. A New Tool in Mass Spectrometric Protein Interaction Analysis. Anal. Chem. 2001, 73, 1927–1934.CrossRefGoogle Scholar
  7. 7.
    Collins, C. J.; Schilling, B.; Young, M. M.; Dollinger, G.; Guy, R. K. Isotopically Labeled Crosslinking Reagents: Resolution of Mass Degeneracy in the Identification of Cross-Linked Peptides. Bioorg. Med. Chem. Lett. 2003, 13, 4023–4026.CrossRefGoogle Scholar
  8. 8.
    Taverner, T.; Hall, N. E.; O’Hair, R. A. J.; Simpson, R. J. Characterization of an Antagonist Interleukin-6 Dimer by Stable Isotope Labeling, Cross-Linking, and Mass Spectrometry. J. Biol. Chem. 2002, 277, 46487–46492.CrossRefGoogle Scholar
  9. 9.
    Bennett, K. L.; Kussmann, M.; Bjork, P.; Godzwon, M.; Mikkelsen, M.; Sorensen, P.; Roepstorff, P. Chemical Cross-Linking with Thiol-cleavable Reagents Combined with Differential Mass Spectrometric Peptide Mapping—A Novel Approach to Assess Intermolecular Protein Contacts. Protein Sci. 2000, 9, 1503–1518.CrossRefGoogle Scholar
  10. 10.
    Sinz, A.; Wang, K. Mapping Protein Interfaces with a Fluorogenic Cross-Linker and Mass Spectrometry: Application to Nebulin-Calmodulin Complexes. Biochemistry. 2001, 40, 7903–7913.CrossRefGoogle Scholar
  11. 11.
    Tang, X.; Munske, G. R.; Siems, W. F.; Bruce, J. E. Mass Spectrometry Identifiable Cross-Linking Strategy for Studying Protein—Protein Interactions. Anal. Chem. 2005, 77, 311–318.CrossRefGoogle Scholar
  12. 12.
    Sinz, A.; Kalkhof, S.; Ihling, C. Mapping Protein Interfaces by a Trifunctional Cross-Linker Combined with MALDI-TOF and ESI-FTICR Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2005, 16, 1921–1931.CrossRefGoogle Scholar
  13. 13.
    Trester-Zedlitz, M.; Kamada, K.; Burley, S. K.; Fenyö, D.; Chait, B. T.; Muir, T. W. A Modular Cross-Linking Approach for Exploring Protein Interactions. J. Am. Chem. Soc. 2003, 125, 2416–2425.CrossRefGoogle Scholar
  14. 14.
    Hurst, G. B.; Lankford, T. K.; Kennel, S. J. Mass Spectrometric Detection of Affinity Purified Crosslinked Peptides. J. Am. Chem. Soc. 2004, 15, 832–839.Google Scholar
  15. 15.
    Comisarow, M. B.; Marshall, A. G. Fourier Transform Ion Cyclotron Resonance Spectroscopy. Chem. Phys. Lett. 1974, 25, 282–283.CrossRefGoogle Scholar
  16. 16.
    Marshall, A. G. Milestones in Fourier Transform Ion Cyclotron Resonance Spectrometry Technique Development. Int. J. Mass Spectrom. 2000, 200, 331–356.CrossRefGoogle Scholar
  17. 17.
    Kruppa, G. H.; Schoeniger, J. S.; Young, M. M. A Top Down Approach to Protein Structural Studies Using Chemical Cross-Linking and Fourier Transform Mass Spectrometry. Rapid Commun. Mass Spectrom. 2003, 17, 155–162.CrossRefGoogle Scholar
  18. 18.
    Novak, P.; Haskins, W. E.; Ayson, M. J.; Jacobsen, R. B.; Schoeniger, J. S.; Leavell, M. D.; Young, M. M.; Kruppa, G. H. Unambiguous Assignment of Intramolecular Chemical Cross-Links in Modified Mammalian Membrane Proteins by Fourier Transform-Tandem Mass Spectrometry. Anal. Chem. 2005, 77, 5101–5108.CrossRefGoogle Scholar
  19. 19.
    Novak, P.; Young, M. M.; Schoeniger, J. S.; Kruppa, G. H. A Top-Down Approach to Protein Structure Studies Using Chemical Cross-Linking and Fourier Transform Mass Spectrometry. Eur. J. Mass Spectrom 2003, 9, 623–631.CrossRefGoogle Scholar
  20. 20.
    Schulz, D. M.; Ihling, C.; Clore, G. M.; Sinz, A. Mapping the Topology and Determination of a Low Resolution Three-Dimensional Structure of the Calmodulin-Melittin Complex by Chemical Cross-Linking and High-Resolution FTICR Mass Spectrometry: Direct Demonstration of Multiple Binding Modes. Biochemistry. 2004, 43, 4703–4715.CrossRefGoogle Scholar
  21. 21.
    Kalkhof, S.; Ihling, C.; Mechtler, K.; Sinz, A. Chemical Cross-Linking and High-Performance Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Protein Interaction Analysis: Application to a Calmodulin/Target Peptide Complex. Anal. Chem. 2005, 77, 495–503.CrossRefGoogle Scholar
  22. 22.
    Crivici, A.; Ikura, M. Molecular and Structural Basis of Target Recognition by Calmodulin. Annu. Rev. Biophys. Biomol. Struct. 1995, 24, 85–116.CrossRefGoogle Scholar
  23. 23.
    Gu, C.; Cooper, D. M. F. Calmodulin Binding Sites on Adenylyl Cyclase VIII. J. Biol. Chem. 1999, 274, 8012–8021.CrossRefGoogle Scholar
  24. 24.
    Bähler, M.; Rhoads, A. Calmodulin Signaling via the IQ Motif. FEBS Lett. 2002, 513, 107–113.CrossRefGoogle Scholar
  25. 25.
    Schmidt, A.; Kalkhof, S.; Ihling, C.; Cooper, D. M. F.; Sinz, A. Mapping Protein Interfaces by Chemical Cross-Linking and FTICR Mass Spectrometry: Application to a Calmodulin/Adenylyl Cyclase 8 Peptide Complex. Eur. J. Mass Spectrom 2005, 11, 524–534.Google Scholar
  26. 26.
    Laemmli, U. K. Cleavage of Structural Proteins During Assembly of Head of Bacteriophage-T4. Nature. 1970, 227, 680–685.CrossRefGoogle Scholar
  27. 27.
    Jensen, O. N.; Shevchenko, A.; Mann, M. In: Protein Structure, A Practical Approach; Creighton, T. E. Ed.; Oxford University Press: Oxford, 1997, p 48.Google Scholar
  28. 28.
    BioAPEX User’s Manual Vol. 1.1; Bruker Daltonics: Billerica, MA, 1996.Google Scholar
  29. 29.
    Peri, S.; Steen, H.; Pandey, A. GPMAW—A Software Tool for Analyzing Proteins and Peptides. Trends Biochem. Sci. 2001, 26, 687–689.CrossRefGoogle Scholar
  30. 30.
    Hermanson G. T. Bioconjugate Techniques; Academic Press Inc.: San Diego, 1996, pp 187–227.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2006

Authors and Affiliations

  • Christian Ihling
    • 1
    • 2
  • Andreas Schmidt
    • 1
    • 2
  • Stefan Kalkhof
    • 1
  • Daniela M. Schulz
    • 1
  • Christoph Stingl
    • 2
  • Karl Mechtler
    • 2
  • Michael Haack
    • 3
  • Annette G. Beck-Sickinger
    • 3
  • Dermot M. F. Cooper
    • 4
  • Andrea Sinz
    • 5
  1. 1.Biotechnological-Biomedical Center, Faculty of Chemistry and MineralogyUniversity of LeipzigLeipzigGermany
  2. 2.Institute of Molecular PathologyViennaAustria
  3. 3.Department of Bioscience, Pharmacy, and Psychology, Institute of BiochemistryUniversity of LeipzigLeipzigGermany
  4. 4.Department of PharmacologyUniversity of CambridgeCambridgeUK
  5. 5.Biotechnological-Biomedical Center, Faculty of Chemistry and MineralogyUniversity of LeipzigLeipzigGermany

Personalised recommendations