title: Chemical cross-linking and high-performance Fourier transform ion cyclotron resonance mass spectrometry for protein interaction analysis: application to a calmodulin/target peptide complex.
abstract: Chemical cross-linking has proved successful in combination with mass spectrometry as a tool for low-resolution structure determination of proteins. The integration of chemical cross-linking with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to determine protein interfaces was tested on the calcium-dependent complex between calmodulin (CaM) and a 26-amino acid peptide derived from the skeletal muscle myosin light chain kinase (M13). Different amine-reactive, homobifunctional cross-linkers and a "zero-length" cross-linker were employed. The covalently attached complexes were separated from nonreacted proteins by one-dimensional gel electrophoresis, and the bands of interest were excised and in-gel digested with trypsin. Digestion of the cross-linked complexes resulted in complicated peptide mixtures, which were analyzed by nano-HPLC/nano-ESI-FTICR mass spectrometry. The distance constraints obtained by chemical cross-linking were in agreement with the published NMR structure of the CaM/M13 complex, pointing to residues Lys-18 and Lys-19 of M13 being cross-linked with the central alpha-helix of CaM. Thus, the integrated approach described herein has proven to be an efficient tool for mapping the topology of the CaM/M13 complex. As such it is applicable as a general strategy for the investigation of the spatial organization of protein complexes and complements existing techniques, such as X-ray crystallography and NMR spectroscopy.
authors.affiliation: Biotechnological-Biomedical Center, Faculty of Chemistry and Mineralogy, University of Leipzig, D-04103 Leipzig, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Kalkhof
authors.forename: Christian
authors.initials: C
authors.lastname: Ihling
authors.forename: Karl
authors.initials: K
authors.lastname: Mechtler
authors.forename: Andrea
authors.initials: A
authors.lastname: Sinz
mesh_terms: D000272:Adipates / Q000737:chemistry; D002147:Calmodulin / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry*; D017357:Cyclotrons; D004591:Electrophoresis, Polyacrylamide Gel; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry; D005583:Fourier Analysis; D013058:Mass Spectrometry / Q000379:methods*; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry*; D036103:Nanotechnology; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry*; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013386:Succinates / Q000737:chemistry; D013388:Succinimides / Q000737:chemistry
chemical_list: D000272:Adipates; D002147:Calmodulin; D003432:Cross-Linking Reagents; C107660:M13 protein (myosin light-chain kinase); D010446:Peptide Fragments; D011506:Proteins; D013386:Succinates; D013388:Succinimides; C516560:disuccinimidyl adipate; C516559:ethyleneglycol bis(sulfosuccinimidyl succinate); C035760:bis(sulfosuccinimidyl)suberate; D009219:Myosin-Light-Chain Kinase; D005022:Ethyldimethylaminopropyl Carbodiimide