Identification of secreted bacterial proteins by noncanonical amino acid tagging

Proc Natl Acad Sci U S A. 2014 Jan 7;111(1):433-8. doi: 10.1073/pnas.1301740111. Epub 2013 Dec 17.


Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins. Selective labeling of microbial proteins is accomplished via translational incorporation of azidonorleucine (Anl), a methionine surrogate that requires a mutant form of the methionyl-tRNA synthetase for activation. Secreted pathogen proteins containing Anl can be tagged by azide-alkyne cycloaddition and enriched by affinity purification. Application of the method to analysis of the type III secretion system of the human pathogen Yersinia enterocolitica enabled efficient identification of secreted proteins, identification of distinct secretion profiles for intracellular and extracellular bacteria, and determination of the order of substrate injection into host cells. This approach should be widely useful for the identification of virulence factors in microbial pathogens and the development of potential new targets for antimicrobial therapy.

Keywords: BONCAT; Yop; click chemistry; proteomics.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acids / chemistry*
  • Anti-Bacterial Agents / chemistry
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism*
  • HeLa Cells
  • Humans
  • Mass Spectrometry
  • Methionine-tRNA Ligase / chemistry
  • Microbiological Techniques*
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Models, Biological
  • Mutation
  • Norleucine / chemistry
  • Proteome
  • Proteomics / methods
  • Time Factors
  • Virulence Factors
  • Yersinia enterocolitica / metabolism


  • Amino Acids
  • Anti-Bacterial Agents
  • Bacterial Proteins
  • Proteome
  • Virulence Factors
  • Norleucine
  • Methionine-tRNA Ligase