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

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1657; p. 361
• Main Authors: Laventie, Benoît-Joseph, Glatter, Timo, Jenal, Urs
• Format: Journal Article
• Language: English
• Published: United States 2017
• Subjects: Bacterial Proteins - metabolism; Carrier Proteins - metabolism; Chromatography, Liquid; Cross-Linking Reagents; Cyclic GMP - analogs & derivatives; Cyclic GMP - metabolism; Databases, Genetic; Mass Spectrometry - methods; Protein Binding; Pseudomonas aeruginosa - metabolism; Tandem Mass Spectrometry; c-di-GMP effector; EAL; PilZ; Capture compound; Pseudomonas aeruginosa; GGDEF; Mass spectrometry; Photoactivable crosslinker
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-7240-1_28

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.