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

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.