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Reference Category Journals
DOI 10.1016/j.jasms.2005.07.020
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Title (Primary) Mapping protein interfaces by a trifunctional cross-linker combined with MALDI-TOF and ESI-FTICR mass spectrometry
Author Sinz, A.; Kalkhof, S.; Ihling, C.
Source Titel Journal of the American Society for Mass Spectrometry
Year 2005
Department PROTEOM
Volume 16
Issue 12
Page From 1921
Page To 1931
Language englisch
Abstract 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-l,3'-dithiopropionate). This trifunctional cross-linker possesses one amine-reactive and one photoreactive site and, additionally, allows an affinity-based enrichment of cross-linker containing species. As a model system, we chose the Ca2+-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
Persistent UFZ Identifier
Sinz, A., Kalkhof, S., Ihling, C. (2005):
Mapping protein interfaces by a trifunctional cross-linker combined with MALDI-TOF and ESI-FTICR mass spectrometry
J. Am. Soc. Mass Spectrom. 16 (12), 1921 - 1931 10.1016/j.jasms.2005.07.020