Photo-cross-linking and high-resolution mass spectrometry for assignment of RNA-binding sites in RNA-binding proteins

Nat Methods. 2014 Oct;11(10):1064-70. doi: 10.1038/nmeth.3092. Epub 2014 Aug 31.


RNA-protein complexes play pivotal roles in many central biological processes. Although methods based on high-throughput sequencing have advanced our ability to identify the specific RNAs bound by a particular protein, there is a need for precise and systematic ways to identify RNA interaction sites on proteins. We have developed an experimental and computational workflow combining photo-induced cross-linking, high-resolution mass spectrometry and automated analysis of the resulting mass spectra for the identification of cross-linked peptides, cross-linking sites and the cross-linked RNA oligonucleotide moieties of such RNA-binding proteins. The workflow can be applied to any RNA-protein complex of interest or to whole proteomes. We applied the approach to human and yeast mRNA-protein complexes in vitro and in vivo, demonstrating its powerful utility by identifying 257 cross-linking sites on 124 distinct RNA-binding proteins. The open-source software pipeline developed for this purpose, RNP(xl), is available as part of the OpenMS project.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / chemistry
  • Automation
  • Binding Sites
  • Computer Simulation
  • Cross-Linking Reagents / chemistry
  • Fungal Proteins / chemistry
  • Humans
  • Mass Spectrometry / methods*
  • Oligonucleotides / chemistry
  • Peptides / chemistry
  • Proteome
  • Proteomics / methods
  • RNA / chemistry*
  • RNA-Binding Proteins / chemistry*
  • Software


  • Amino Acids
  • Cross-Linking Reagents
  • Fungal Proteins
  • Oligonucleotides
  • Peptides
  • Proteome
  • RNA-Binding Proteins
  • RNA