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Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states
- Source :
- eLife, Vol 5 (2016)
- Publication Year :
- 2016
- Publisher :
- eLife Sciences Publications Ltd, 2016.
-
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.
Details
- Language :
- English
- ISSN :
- 2050084X
- Volume :
- 5
- Database :
- Directory of Open Access Journals
- Journal :
- eLife
- Publication Type :
- Academic Journal
- Accession number :
- edsdoj.1b4dc53824b43dfb9c0301bb08e8ae7
- Document Type :
- article
- Full Text :
- https://doi.org/10.7554/eLife.12509