Mapping Protein Interfaces by a Trifunctional Cross-Linker Combined with MALDI-TOF and ESI-FTICR Mass Spectrometry

• Published in: Journal of the American Society for Mass Spectrometry Vol. 16; no. 12; pp. 1921 - 1931
• Main Authors: Sinz, Andrea, Kalkhof, Stefan, Ihling, Christian
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Inc 01.12.2005; Elsevier Science; Springer Nature B.V
• Subjects: Analytical, structural and metabolic biochemistry; Beads; Binding Sites; Biological and medical sciences; Calcium - chemistry; Calmodulin; Calmodulin - analysis; Calmodulin - chemistry; Cross-Linking Reagents - chemistry; Crosslinking; Cyclotron resonance; Cyclotrons; Desorption; Enrichment; Fourier transforms; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; High performance liquid chromatography; Ionization; Ions; Lasers; Mapping; Mass spectrometry; Myosin; Myosin-Light-Chain Kinase - analysis; Myosin-Light-Chain Kinase - chemistry; Peptide Fragments - analysis; Peptide Fragments - chemistry; Peptides; Protein Binding; Protein Interaction Mapping - methods; Proteins; Reaction products; Scientific imaging; Spectrometry, Mass, Electrospray Ionization - methods; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Spectroscopy; Spectroscopy, Fourier Transform Infrared - methods; Fourier transformation; Biotinylation; Time of flight method; Crosslink agent; Matrix assisted laser desorption ionization; Ion cyclotron resonance spectrometry; Mass spectrometry; Electrospray; Interface; Protein
• ISSN: 1044-0305; 1879-1123
• DOI: 10.1016/j.jasms.2005.07.020

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.