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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
Articles

Abstract

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.

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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

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