Identification and quantification of low-molecular weight bacterial nucleotide signaling molecules by HPLC-coupled mass spectrometry including ion mobility technology

• Applicant: Professor Dr. Roland Seifert, since 7/2019
• Subject Area: Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology ; Analytical Chemistry
• Term: from 2016 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 314721994

Numerous low-molecular weight bacterial signaling molecules, either present in the cytosol or being secreted, have been described during the last years. Among them, linear as well as cyclic nucleotides play major roles as second messengers in the regulation of important bacterial functions, i.e. guanosine 3´-diphosphate, 5´-triphosphate (pppGpp), guanosine-3´,5´-bispyrophosphate (ppGpp), 3´,5´-cyclic adenosine monophosphate (cAMP) and 3´,5´-cyclic guanosine monophosphate (cGMP), cyclic dimeric guanosine 3´,5´-monophosphate (c-di-GMP), cyclic dimeric adenosine 3´,5´-monophosphate (c-di-AMP) and cyclic 3´,5´-guanosine monophosphate-3´,5´-adenosine monophosphate (3´,3´-cGAMP). Due to their low physiological concentrations and rapid metabolism, identification and quantification of these molecules is a highly challenging analytical task. We have developed sensitive and specific HPLC-coupled tandem mass spectrometry (LC-MS/MS) methods with the main focus on unequivocally quantification of linear and cyclic nucleotides, cyclic dinucleotides, and their respective degradation products. In this case, LC-MS/MS with inclusion of isotope-labeled internal standards and analysis of specific quantifier and several qualifier mass transitions represents the analytical method of choice. However, special care has to be taken regarding the initial sample preparation steps, the robustness of the HPLC methods, and a reliable MS/MS procedure. The established procedures can easily be adapted for the analyses of additional molecules, e.g. nucleotide analogs. It is intended in this project to further optimize the already established LC-MS/MS methods with respect to further improvement of the sample extraction procedures, establishment of specific extraction procedures for secreted signaling molecules from cellular supernatants, and biological syntheses of isotope-labeled internal standards by using recombinant enzymes. Our expertise in LC-MS/MS technology will be provided in close collaboration with many partners within the SPP 1879 as a Z unit, especially addressing the identification and quantification of bacterial nucleotide signaling molecules.

• DFG Programme: Priority Programmes
• Subproject of: SPP 1879: Nucleotide Second Messenger Signaling in Bacteria
• Co-Investigator: Dr. Heike Bähre
• Ehemaliger Antragsteller: Professor Dr. Volkhard Kaever, until 7/2019