Specifying RNA-Binding Regions in Proteins by Peptide Cross-Linking and Affinity Purification

J Proteome Res. 2017 Aug 4;16(8):2762-2772. doi: 10.1021/acs.jproteome.7b00042. Epub 2017 Jul 7.


RNA-binding proteins (RBPs) allow cells to carry out pre-RNA processing and post-transcriptional regulation of gene expression, and aberrations in RBP functions have been linked to many diseases, including neurological disorders and cancer. Human cells encode thousands of RNA-binding proteins with unique RNA-binding properties. These properties are regulated through modularity of a large variety of RNA-binding domains, rendering RNA-protein interactions difficult to study. Recently, the introduction of proteomics methods has provided novel insights into RNA-binding proteins at a systems level. However, determining the exact protein sequence regions that interact with RNA remains challenging and laborious, especially considering that many RBPs lack canonical RNA-binding domains. Here we describe a streamlined proteomic workflow called peptide cross-linking and affinity purification (pCLAP) that allows rapid characterization of RNA-binding regions in proteins. pCLAP is based upon the combined use of UV cross-linking and enzymatic digestion of RNA-bound proteins followed by single-shot mass spectrometric analysis. To benchmark our method, we identified the binding regions for polyadenylated RNA-binding proteins in HEK293 cells, allowing us to map the mRNA interaction regions of more than 1000 RBPs with very high reproducibility from replicate single-shot analyses. Our results show specific enrichment of many known RNA-binding regions on many known RNA-binding proteins, confirming the specificity of our approach.

Keywords: RNA-binding proteins; RNA-binding regions; mass spectrometry; single-shot MS analysis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites*
  • Chromatography, Affinity
  • HEK293 Cells
  • Humans
  • Methods
  • Peptides / metabolism*
  • Protein Binding
  • Proteomics / methods*
  • RNA, Messenger / metabolism
  • RNA-Binding Proteins / analysis
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / metabolism*
  • Reproducibility of Results
  • Workflow


  • Peptides
  • RNA, Messenger
  • RNA-Binding Proteins