Spectrophotometric and mass spectroscopic methods for the quantification and kinetic evaluation of in vitro c-di-GMP synthesis

Abstract

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.