title: Capture compound mass spectrometry--a powerful tool to identify novel c-di-GMP effector proteins.
abstract: Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail. We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms(1). Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.
authors: Focal Area Infection Biology, Biozentrum of the University of Basel.
authors: Benoît-Joseph
authors: BJ
authors: Laventie
authors: Focal Area Infection Biology, Biozentrum of the University of Basel.
authors: Jutta
authors: J
authors: Nesper
authors: Proteomics Core Facility, Biozentrum of the University of Basel.
authors: Erik
authors: E
authors: Ahrné
authors: Proteomics Core Facility, Biozentrum of the University of Basel.
authors: Timo
authors: T
authors: Glatter
authors: Proteomics Core Facility, Biozentrum of the University of Basel.
authors: Alexander
authors: A
authors: Schmidt
authors: Focal Area Infection Biology, Biozentrum of the University of Basel; urs.jenal@unibas.ch.
authors: Urs
authors: U
authors: Jenal
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry*; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D019761:Phosphorus-Oxygen Lyases / Q000737:chemistry / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C062025:bis(3',5')-cyclic diguanylic acid; D019761:Phosphorus-Oxygen Lyases; C060720:diguanylate cyclase; D006152:Cyclic GMP