Analysis of Nucleotide Pools in Bacteria Using HPLC-MS in HILIC Mode

Nucleotides, nucleosides and their derivatives are present in cells at varying concentrations that rapidly change with the nutritional, and energetic status of the cell. Knowledge of the concentration dynamics of these molecules is instrumental for understanding their regulatory effects. Determination of these concentrations 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 to determine 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. * (c-di-AMP) : cyclic diadenylate (c-di-GMP) : cyclic diguanylate ApxN : dinucleoside polyphosphates (pppGpp) : guanosine pentaphosphate (ppGpp) : guanosine tetraphosphate (LB) : lysogeny broth (SR) : stringent response (AEC) : Adenylate Energy Charge