Mapping protein interfaces by a trifunctional cross-linker combined with MALDI-TOF and ESI-FTICR mass spectrometry.
Chemical cross-linking of protein complexes has gained renewed interest in combination with mass spectrometric analysis of the reaction products as it allows a rapid mapping of protein interfaces, which is crucial for understanding protein/protein interactions. The identification of cross-linking products from the complex mixtures created after the cross-linking reaction, however, remains a daunting task. To facilitate the identification of cross-linking products, we explore the use of the commercially available biotinylated cross-linking reagent sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]ethyl-1,3'-dithiopropionate). This trifunctional cross-linker possesses one amine-reactive and one photo-reactive site and, additionally, allows an affinity-based enrichment of cross-linker containing species. As a model system, we chose the Ca(2+)-dependent complex between calmodulin and its target peptide M13, which represents a part of the C-terminal sequence of the skeletal muscle myosin light chain kinase. After the cross-linking reaction, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and one-dimensional gel electrophoresis were employed to check for the extent of cross-linking product formation. The cross-linking reaction mixtures were subjected to tryptic in-solution digestion. Biotinylated peptides, e.g., peptides that had been modified by the cross-linker as well as cross-linked peptides, were enriched on monomeric avidin beads after several washing steps had been performed. Peptide mixtures were analyzed by MALDI-TOFMS, nano-high-performance liquid chromatography (HPLC)/nano-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS), and tandem MS. We demonstrate that an enrichment of cross-linker containing species allows a more efficient identification of interacting amino acid sequences in protein complexes. This strategy is expected to be especially beneficial for investigating large protein assemblies.
Sinz
Andrea
A
Biotechnological-Biomedical Center, Faculty of Chemistry and Mineralogy, University of Leipzig, Germany. sinz@chemie.uni-leipzig.de
Kalkhof
Stefan
S
Ihling
Christian
C
D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D002147:Calmodulin / Q000032:analysis / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D017357:Cyclotrons; D009219:Myosin-Light-Chain Kinase / Q000032:analysis / Q000737:chemistry*; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
D002147:Calmodulin; D003432:Cross-Linking Reagents; C107660:M13 protein (myosin light-chain kinase); D010446:Peptide Fragments; D009219:Myosin-Light-Chain Kinase; D002118:Calcium