Simplified Protocol for Cross-linking Mass Spectrometry Using the MS-Cleavable Cross-linker DSBU with Efficient Cross-link Identification
- Dongqing Pan*Dongqing Pan*E-mail: [email protected]Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, GermanyMore by Dongqing Pan
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- Andreas BrockmeyerAndreas BrockmeyerDepartment of Chemical Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, GermanyMore by Andreas Brockmeyer
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- Franziska MuellerFranziska MuellerDepartment of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, GermanyMore by Franziska Mueller
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- Andrea MusacchioAndrea MusacchioDepartment of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, GermanyCentre for Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Universitaetsstrasse, 45141 Essen, GermanyMore by Andrea Musacchio
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- Tanja Bange*Tanja Bange*E-mail: [email protected]Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, GermanyDepartment for Systems Chronobiology, Institute of Medical Psychology, LMU Munich, Goethe-Str. 31, 80336 Munich, GermanyMore by Tanja Bange
Abstract
Chemical cross-linking combined with mass spectrometry (MS) is a powerful approach to identify and map protein–protein interactions. Its applications support computational modeling of three-dimensional structures and complement classical structural methodologies such as X-ray crystallography, NMR spectroscopy, and electron microscopy (EM). A plethora of cross-linkers, MS methods, and data analysis programs have been developed, but due to their methodological complexity application is currently reserved for specialized mass spectrometry laboratories. Here, we present a simplified single-step purification protocol that results in improved identifications of cross-linked peptides. We describe an easy-to-follow pipeline that combines the MS-cleavable cross-linker DSBU (disuccinimidyl dibutyric urea), a Q-Exactive mass spectrometer, and the dedicated software MeroX for data analysis to make cross-linking MS accessible to structural biology and biochemistry laboratories. In experiments focusing on kinetochore subcomplexes containing 4–10 subunits (so-called KMN network), one-step peptide purification, and enrichment by size-exclusion chromatography yielded identification of 135–228 non-redundant cross-links (577–820 cross-linked peptides) from each experiment. Notably, half of the non-redundant cross-links identified were not lysine–lysine cross-links and involved side chains with hydroxy groups. The new pipeline has a comparable potential toward the identification of protein–protein interactions as previously used pipelines based on isotope-labeled cross-linkers. A newly identified cross-link enabled us to improve our 3D-model of the KMN, emphasizing the power of cross-linking data for evaluation of low-resolution EM maps. In sum, our optimized experimental scheme represents a viable shortcut toward obtaining reliable cross-link data sets.
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