Simplified Protocol for Cross-linking Mass Spectrometry Using the MS-Cleavable Cross-linker DSBU with Efficient Cross-link Identification
2018
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 s...
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