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c-di-GMP Signaling pp 361–376Cite as

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

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Part of the book series: Methods in Molecular Biology ((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.

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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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Authors and Affiliations

  1. Infection Biology, Biozentrum, University of Basel, Basel, Switzerland

    Benoît-Joseph Laventie & Urs Jenal

  2. Proteomics Core Facility, Biozentrum, University of Basel, Basel, Switzerland

    Timo Glatter

  3. Facility for Mass Spectrometry and Proteomics, Max-Planck Institute for Terrestrial Microbiology, Marburg, Germany

    Timo Glatter

Authors
  1. Benoît-Joseph Laventie
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  2. Timo Glatter
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  3. Urs Jenal
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Corresponding author

Correspondence to Benoît-Joseph Laventie .

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Editors and Affiliations

  1. Department of Biological Sciences, Binghamton University Department of Biological Sciences, Binghamton, New York, USA

    Karin Sauer

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Laventie, BJ., Glatter, T., Jenal, U. (2017). Pull-Down with a c-di-GMP-Specific Capture Compound Coupled to Mass Spectrometry as a Powerful Tool to Identify Novel Effector Proteins. In: Sauer, K. (eds) c-di-GMP Signaling. Methods in Molecular Biology, vol 1657. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7240-1_28

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  • DOI: https://doi.org/10.1007/978-1-4939-7240-1_28

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7239-5

  • Online ISBN: 978-1-4939-7240-1

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