Isotope-labeled cross-linkers and Fourier transform ion cyclotron resonance mass spectrometry for structural analysis of a protein/peptide complex.
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 alpha-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.
Ihling
Christian
C
Biotechnological-Biomedical Center, Faculty of Chemistry and Mineralogy, University of Leipzig, Leipzig, Germany.|Institute of Molecular Pathology, Vienna, Austria.
Schmidt
Andreas
A
Biotechnological-Biomedical Center, Faculty of Chemistry and Mineralogy, University of Leipzig, Leipzig, Germany.|Institute of Molecular Pathology, Vienna, Austria.
Kalkhof
Stefan
S
Biotechnological-Biomedical Center, Faculty of Chemistry and Mineralogy, University of Leipzig, Leipzig, Germany.
Schulz
Daniela M
DM
Biotechnological-Biomedical Center, Faculty of Chemistry and Mineralogy, University of Leipzig, Leipzig, Germany.
Stingl
Christoph
C
Institute of Molecular Pathology, Vienna, Austria.
Mechtler
Karl
K
Institute of Molecular Pathology, Vienna, Austria.
Haack
Michael
M
Department of Bioscience, Pharmacy, and Psychology, Institute of Biochemistry, University of Leipzig, Leipzig, Germany.
Beck-Sickinger
Annette G
AG
Department of Bioscience, Pharmacy, and Psychology, Institute of Biochemistry, University of Leipzig, Leipzig, Germany.
Cooper
Dermot M F
DMF
Department of Pharmacology, University of Cambridge, Cambridge, UK.
Sinz
Andrea
A
Biotechnological-Biomedical Center, Faculty of Chemistry and Mineralogy, University of Leipzig, Linnéstrasse 3, D-04103, Leipzig, Germany. sinz@chemie.uni-leipzig.de.
D000262:Adenylyl Cyclases / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D001923:Brain Chemistry; D002147:Calmodulin / Q000737:chemistry*; D003432:Cross-Linking Reagents; D017357:Cyclotrons; D007553:Isotope Labeling / Q000379:methods; D013058:Mass Spectrometry; D010449:Peptide Mapping / Q000379:methods*; D011485:Protein Binding; D020539:Sequence Analysis, Protein / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D013552:Swine
D002147:Calmodulin; D003432:Cross-Linking Reagents; D000262:Adenylyl Cyclases