Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states
- Peking Union Medical College, Chinese Academy of Medical Sciences, China
- National Institute of Biological Sciences, China
- Institute of Computing Technology, Chinese Academy of Sciences, China
- Tsinghua University, China
- Chinese Academy of Medical Sciences, Peking Union Medical College, China
- Tianjin University, China
Abstract
To improve chemical cross-linking of proteins coupled with mass spectrometry (CXMS), we developed a lysine-targeted enrichable cross-linker containing a biotin tag for affinity purification, a chemical cleavage site to separate cross-linked peptides away from biotin after enrichment, and a spacer arm that can be labeled with stable isotopes for quantitation. By locating the flexible proteins on the surface of 70S ribosome, we show that this trifunctional cross-linker is effective at attaining structural information not easily attainable by crystallography and electron microscopy. From a crude Rrp46 immunoprecipitate, it helped identify two direct binding partners of Rrp46 and 15 protein-protein interactions (PPIs) among the co-immunoprecipitated exosome subunits. Applying it to E. coli and C. elegans lysates, we identified 3130 and 893 inter-linked lysine pairs, representing 677 and 121 PPIs. Using a quantitative CXMS workflow we demonstrate that it can reveal changes in the reactivity of lysine residues due to protein-nucleic acid interaction.
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© 2016, Tan et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states
eLife 5:e12509.
https://doi.org/10.7554/eLife.12509
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