A liquid chromatography-coupled tandem mass spectrometry method for quantitation of cyclic di-guanosine monophosphate.
Cyclic di-guanosine monophosphate (c-di-GMP) represents an important ubiquitous second messenger in bacteria. It controls the transition between a sessile and a motile lifestyle of bacteria and, hence, affects the formation of biofilms which are highly resistant to antimicrobial treatment. c-di-GMP is synthesized by di-guanylate cyclases (DGCs) and degraded by specific phosphodiesterases (PDEs), two highly abundant protein families in bacteria. We have established a robust and highly sensitive high performance liquid chromatography-coupled tandem mass spectrometry (HPLC-MS/MS) based method for the quantitation of c-di-GMP and investigated various method performance parameters such as limit of detection (LOD), lower limit of quantitation (LLOQ), linearity, accuracy, recovery and analyte stability. As a proof of principle we used this method to accurately measure the activity of the prototype DGC PleD* from Caulobacter crescentus in vitro. In addition the methodology was successfully applied to determine in vivo levels of c-di-GMP in bacterial extracts of E. coli at different stages of bacterial growth. This demonstrates that our method is suitable for the sensitive and specific quantitation of c-di-GMP in bacterial cell extracts.
Spangler
Christian
C
Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany. spangler.christian@mh-hannover.de
Böhm
Alex
A
Jenal
Urs
U
Seifert
Roland
R
Kaever
Volkhard
V
D016935:Caulobacter crescentus / Q000201:enzymology; D002853:Chromatography, Liquid / Q000379:methods; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000032:analysis; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D019761:Phosphorus-Oxygen Lyases / Q000378:metabolism; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
D029968:Escherichia coli Proteins; C062025:bis(3',5')-cyclic diguanylic acid; D019761:Phosphorus-Oxygen Lyases; C060720:diguanylate cyclase; D006152:Cyclic GMP