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

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.