Mapping protein interfaces by chemical cross-linking and FTICR mass spectrometry: application to a calmodulin / adenylyl cyclase 8 peptide complex

Eur. J. Mass Spectrom. 11, 525–534 (2005)
DOI: 10.1255/ejms.748

Mapping protein interfaces by chemical cross-linking and FTICR mass spectrometry: application to a calmodulin / adenylyl cyclase 8 peptide complex

Andreas Schmidt,^a Stefan Kalkhof,^a Christian Ihling,^a Dermot M.F. Cooper,^b and Andrea Sinz^a,*
^a Biotechnological-Biomedical Center, Faculty of Chemistry and Mineralogy, University of Leipzig, D-04103 Leipzig, Germany
^bDepartment of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, UK

ABSTRACT:

Chemical cross-linking – an established technique in protein chemistry – has reemerged, in combination with mass spectrometric analysis of the reaction products, as a valuable tool to identify interacting amino acid sequences in protein complexes. In the present study, we are mapping the interface of the calcium-dependent complex between calmodulin (CaM) and a peptide derived from the C-terminal region of adenylyl cyclase 8 (AC 8). Cross-linking reactions are performed using the two amine-reactive, isotope-labeled (d₀ and d₄) cross-linkers BS³ (bis[sulfosuccinimidyl]suberate) and BS²G (disulfosuccinimidyl glutarate) as well as the ‘zero-length’ cross-linker (EDC, ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride). After separation of the cross-linking reaction mixtures by onedimensional gel electrophoresis (SDS-PAGE) and in-gel digestion of the cross-linked complexes, the resulting peptide mixtures are analyzed by nano-HPLC/ nano-ESI/FT-ICR/MS (nano-high-performance liquid chromatography/nano-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry). The identified intermolecular cross-linking products will give further insight into calmodulin/adenylyl cyclase 8 interaction.

Keywords: chemical cross-linking; protein/protein interaction; protein conformation; FTICR mass spectrometry; nano-HPLC; tryptic digestion; isotope-labeled cross-linker; adenylyl cyclase; calmodulin

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