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Pull-Down with a c-di-GMP-Specific Capture Compound Coupled to Mass Spectrometry as a Powerful Tool to Identify Novel Effector Proteins

  • Benoît-Joseph Laventie
  • Timo Glatter
  • Urs Jenal
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1657)

Abstract

Capture compound technology coupled to mass spectrometry (CCMS) allows to biochemically identify ligand receptors. Using a c-di-GMP-specific Capture Compound, we adapted this method for the identification and characterization of c-di-GMP binding proteins in any bacterial species. Because in silico analysis often fails to predict novel c-di-GMP effectors, this universal method aims at better defining the cellular c-di-GMP network in a wide range of bacteria. CCMS was successfully applied in several bacterial species (Nesper et al., J Proteom 75:4874–4878, 2012; Steiner et al., EMBO J 32:354–368, 2013; Tschowri et al., Cell 158:1136–1147, 2014; Trampari et al., J Biol Chem 290:24470–24483, 2015; Rotem et al., J Bacteriol 198:127–137, 2015). To outline the detailed protocol and to illustrate its power, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control, as an example. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network.

Key words

Capture compound Photoactivable crosslinker Mass spectrometry c-di-GMP effector EAL GGDEF PilZ Pseudomonas aeruginosa 

Notes

Acknowledgments

We thank Jutta Nesper who established the protocol and Alberto Reinders for his work in adapting the CCMS conditions for P. aeruginosa. We also thank Pablo Manfredi for the annotation of the P. aeruginosa proteins and Erik Ahrné for his help with the MS data evaluation. This work was supported by the Swiss National Science Foundation (SNF) Sinergia grant CRSII3_127433.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Benoît-Joseph Laventie
    • 1
  • Timo Glatter
    • 2
    • 3
  • Urs Jenal
    • 1
  1. 1.Infection Biology, BiozentrumUniversity of BaselBaselSwitzerland
  2. 2.Proteomics Core Facility, BiozentrumUniversity of BaselBaselSwitzerland
  3. 3.Facility for Mass Spectrometry and ProteomicsMax-Planck Institute for Terrestrial MicrobiologyMarburgGermany

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