期刊
NATURE PROTOCOLS
卷 9, 期 1, 页码 120-137出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2013.168
关键词
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资金
- European Union [HEALTH-F4-2008-201648]
- European Research Council Advanced Grant Proteomics v3.0 of the European Union [233226]
Chemical cross-linking in combination with LC-MS/MS (XL-MS) is an emerging technology to obtain low-resolution structural (distance) restraints of proteins and protein complexes. These restraints can also be used to characterize protein complexes by integrative modeling of the XL-MS data, either in combination with other types of structural information or by themselves, to establish spatial relationships of subunits in protein complexes. Here we present a protocol that has been successfully used to generate XL-MS data from a multitude of native proteins and protein complexes. It includes the experimental steps for performing the cross-linking reaction using disuccinimidyl suberate (a homobifunctional, lysine-reactive cross-linking reagent), the enrichment of cross-linked peptides by peptide size-exclusion chromatography (SEC; to remove smaller, non-cross-linked peptides), instructions for tandem MS analysis and the analysis of MS data via the open-source computational software pipeline xQuest and xProphet (available from http://proteomics.ethz.ch). Once established, this robust protocol should take similar to 4 d to complete, and it is generally applicable to purified proteins and protein complexes.
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