Nucleotides, nucleosides and their derivatives are present in all cells at varying concentrations that change with the nutritional, and energetic status of the cell. Precise measurement of the concentrations of these molecules is instrumental for understanding their regulatory effects. Such measurement is challenging due to the inherent instability of these molecules and, despite many decades of research, the reported values differ widely. Here, we present a comprehensive and easy-to-use approach for determination of the intracellular concentrations of >25 target molecular species. The approach uses rapid filtration and cold acidic extraction followed by high performance liquid chromatography (HPLC) in the hydrophilic interaction liquid chromatography (HILIC) mode using zwitterionic columns coupled with UV and MS detectors. The method reliably detects and quantifies all the analytes expected to be observed in the bacterial cell and paves the way for future studies correlating their concentrations with biological effects.

Department of Molecular Biology Umeå University Building 6K 6L University Hospital Area SE 901 87 Umeå Sweden; Laboratory for Molecular Infection Medicine Sweden Umeå University Building 6K and 6L University Hospital Area 90187 Umeå Sweden; University of Tartu Institute of Technology 50411 Tartu Estonia

Institute of Microbiology Czech Academy of Sciences v v i Vídeňská 1083 142 20 Prague 4 Czech Republic

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic v v i Flemingovonam 2 CZ 166 10 Prague 6 Czech Republic

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic v v i Flemingovonam 2 CZ 166 10 Prague 6 Czech Republic; Charles University Faculty of Science Department of Analytical Chemistry Hlavova 8 128 43 Prague 2 Czech Republic

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The mycobacterial guaB1 gene encodes a guanosine 5'-monophosphate reductase with a cystathionine-β-synthase domain