Chemical Cross-Linking and High-Performance Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Protein Interaction Analysis:  Application to a Calmodulin/Target Peptide Complex

2005 ◽  
Vol 77 (2) ◽  
pp. 495-503 ◽  
Author(s):  
Stefan Kalkhof ◽  
Christian Ihling ◽  
Karl Mechtler ◽  
Andrea Sinz
Keyword(s):  
Mass Spectrometry ◽  
Fourier Transform ◽  
Protein Interaction ◽  
Cyclotron Resonance ◽  
High Performance ◽  
Interaction Analysis ◽  
Cross Linking ◽  
Ion Cyclotron Resonance ◽  
Analysis Application ◽  
Chemical Cross Linking

Monitoring Conformational Changes in Protein Complexes Using Chemical Cross-Linking and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: The Effect of Calcium Binding on the Calmodulin—Melittin Complex

2007 ◽  
Vol 13 (4) ◽  
pp. 281-290 ◽  
Author(s):  
Petr Novak ◽  
Vladimir Havlicek ◽  
Peter J. Derrick ◽  
Kyle A. Beran ◽  
Sajid Bashir ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Fourier Transform ◽  
Conformational Changes ◽  
Calcium Binding ◽  
Cyclotron Resonance ◽  
Structural Changes ◽  
Cross Linking ◽  
Ion Cyclotron Resonance ◽  
Ion Cyclotron ◽  
Chemical Cross Linking

Calmodulin is an EF hand calcium binding protein. Its binding affinities to various protein/peptide targets often depend on the conformational changes induced by the binding of calcium. One such target is melittin, which binds tightly to calmodulin in the presence of calcium, and inhibits its function. Chemical cross-linking combined with Fourier transform ion cyclotron resonance mass spectrometry has been employed to investigate the coordination of calmodulin and melittin in the complex at different concentrations of calcium. This methodology can be used to monitor structural changes in proteins induced by ligand binding and to study the effects these changes have on non-covalent interactions between proteins. Cross-linking results indicate that the binding place of the first melittin in the calcium-free calmodulin form is the same as in the calcium-loaded calmodulin/melittin complex.

Mapping Protein Interfaces by Chemical Cross-Linking and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: Application to a Calmodulin/Adenylyl Cyclase 8 Peptide Complex

2005 ◽  
Vol 11 (5) ◽  
pp. 525-534 ◽  
Author(s):  
Andreas Schmidt ◽  
Stefan Kalkhof ◽  
Christian Ihling ◽  
Dermot M.F. Cooper ◽  
Andrea Sinza
Keyword(s):  
Mass Spectrometry ◽  
Fourier Transform ◽  
Adenylyl Cyclase ◽  
Cyclotron Resonance ◽  
Cross Linking ◽  
Ion Cyclotron Resonance ◽  
Ion Cyclotron ◽  
The Cross ◽  
Adenylyl Cyclase 8 ◽  
Chemical Cross Linking

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 BS3 ( bis[sulfosuccinimidyl]suberate) and BS2G ( bis[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.

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