Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states

Elife. 2016 Mar 8;5:e12509. doi: 10.7554/eLife.12509.


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.

Keywords: 70S ribosome; c. elegans; e. coli; biophysics; cross-linking; exosome; mass spectrometry; protein structure; protein-protein interactions; structural biology.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Caenorhabditis elegans / chemistry
  • Caenorhabditis elegans / physiology
  • Caenorhabditis elegans Proteins / analysis
  • Caenorhabditis elegans Proteins / chemistry
  • Cross-Linking Reagents / metabolism*
  • Escherichia coli / chemistry
  • Escherichia coli / physiology
  • Escherichia coli Proteins / analysis
  • Escherichia coli Proteins / chemistry
  • Protein Conformation
  • Protein Interaction Mapping / methods*
  • Protein Interaction Maps*
  • Ribosomes / chemistry


  • Caenorhabditis elegans Proteins
  • Cross-Linking Reagents
  • Escherichia coli Proteins

Grant support

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.