Mapping protein interfaces by chemical cross-linking and Fourier transform ion cyclotron resonance mass spectrometry: application to a calmodulin / adenylyl cyclase 8 peptide complex.
Chemical cross-linking--an established technique in protein chemistry--has re-emerged, 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 BS(3) (bis[sulfosuccinimidyl]suberate) and BS(2)G (bi[sulfosuccinimidyl] 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 one-dimensional gel electrophoresis (sodium dodecyl sulphate polyacrylamide gel) and in-gel digestion of the cross-linked complexes, the resulting peptide mixtures are analyzed by 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.
Schmidt
Andreas
A
Biotechnological-Biomedical Center, Faculty of Chemistry and Mineralogy, University of Leipzig, D-04103 Leipzig, Germany.
Kalkhof
Stefan
S
Ihling
Christian
C
Cooper
Dermot M F
DM
Sinz
Andrea
A
D000262:Adenylyl Cyclases / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000737:chemistry*; D002417:Cattle; D003432:Cross-Linking Reagents; D017357:Cyclotrons; D005583:Fourier Analysis; D066298:In Vitro Techniques; D007477:Ions; D013058:Mass Spectrometry / Q000379:methods*; D046912:Multiprotein Complexes; D036103:Nanotechnology; D010446:Peptide Fragments / Q000737:chemistry
D002147:Calmodulin; D003432:Cross-Linking Reagents; D007477:Ions; D046912:Multiprotein Complexes; D010446:Peptide Fragments; D000262:Adenylyl Cyclases