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Anal Biochem. 2009 Mar 1;386(1):53-8. doi: 10.1016/j.ab.2008.12.013. Epub 2008 Dec 24.

Quantitative determination of cyclic diguanosine monophosphate concentrations in nucleotide extracts of bacteria by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

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Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden.


The physiological response to small molecules (secondary messengers) is the outcome of a delicate equilibrium between biosynthesis and degradation of the signal. Cyclic diguanosine monophosphate (c-di-GMP) is a novel secondary messenger present in many bacteria. It has a complex cellular metabolism whereby usually more than one enzyme synthesizing and degrading c-di-GMP is encoded by a bacterial genome. To assess the in vivo conditions of c-di-GMP signaling, we developed a high-performance liquid chromatography (HPLC)-mass spectrometry-based method to detect c-di-GMP with high sensitivity and to quantify the c-di-GMP concentration in the bacterial cell as described here in detail. We successfully used the methodology to determine and compare the c-di-GMP concentrations in bacterial species such as Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, and Vibrio cholerae. We describe the use of the methodology to assess the change in c-di-GMP concentration during the growth phase and the contribution of a point mutation in S. typhimurium to the overall cellular c-di-GMP concentration.

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