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.

化学交联结合质谱（Mass Spectrometry，MS）是一种用于鉴定并绘制蛋白质-蛋白质相互作用图谱的有效方法。该方法可辅助三维结构的计算建模，并可作为X射线晶体学（X-ray crystallography）、核磁共振波谱法（NMR spectroscopy）与电子显微镜（Electron Microscopy，EM）等经典结构研究手段的补充。目前已开发出大量交联剂、质谱分析方法与数据分析软件，但由于其方法学复杂度较高，目前仅能在专业的质谱实验室中开展应用。本研究提出一种简化的单步纯化方案，可提升交联肽段的鉴定效率。我们介绍了一套易于操作的分析流程，该流程将可被质谱裂解的交联剂DSBU（disuccinimidyl dibutyric urea）、Q-Exactive质谱仪与专用数据分析软件MeroX相结合，使交联质谱技术可被结构生物学与生物化学实验室所采用。在针对含有4~10个亚基的动粒（kinetochore）亚复合物（即所谓KMN网络）的实验中，通过单步肽段纯化与尺寸排阻色谱（size-exclusion chromatography）富集，单次实验即可鉴定得到135~228条非冗余交联（对应577~820条交联肽段）。值得注意的是，所鉴定得到的非冗余交联中有半数并非赖氨酸-赖氨酸交联，而是涉及带有羟基的侧链。该新型分析流程在蛋白质-蛋白质相互作用鉴定方面的潜力，与此前基于同位素标记交联剂的分析流程相当。一条新鉴定得到的交联位点帮助我们优化了KMN网络的三维模型，凸显了交联数据在评估低分辨率电子显微镜图谱中的应用价值。综上，我们优化的实验方案为获取可靠的交联数据集提供了一条切实可行的捷径。