Spectrophotometric and Mass Spectroscopic Methods for the Quantification and Kinetic Evaluation of In Vitro c-di-GMP Synthesis.

The expression and activity of diguanylate cyclase (DGC) and phosphodiesterase (PDE) enzymes are responsible for modulating and maintaining the intracellular concentration of the bacterial second messenger cyclic diguanosine-monophosphate (c-di-GMP). Here, we describe an in vitro method for the spectrophotometric detection and quantification of DGC catalyzed c-di-GMP synthesis through adaptation of the EnzChek ^® Pyrophosphate Assay Kit. We also outline a method for the quantification of c-di-GMP produced in this in vitro reaction using Ultra-Performance Liquid Chromatography tandem Mass Spectrometry (UPLC-MS/MS). These methods can be leveraged for a number of experimental applications including the evaluation of enzyme activity for the in vitro synthesis of c-di-GMP, examination of how molecular signals impact these activities, identifying the catalytic properties of hybrid DGC-PDE proteins, and the development of DGC inhibitors.