論文

査読有り
2018年8月

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

Analytical chemistry
  • Pan D
  • ,
  • Brockmeyer A
  • ,
  • Mueller F
  • ,
  • Musacchio A
  • ,
  • Bange T

DOI
10.1021/acs.analchem.8b02593

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.

リンク情報
DOI
https://doi.org/10.1021/acs.analchem.8b02593
PubMed
https://www.ncbi.nlm.nih.gov/pubmed/30074391
URL
http://orcid.org/0000-0003-3701-8442