Determination of modified nucleosides in Escherichia coli by Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry

To date, there are about 100 modified nucleosides (MNs) have been identified in RNA species and the majority of them were found in tRNAs. Except their common properties as components of RNAs, MNs have been confirmed to be of multifunctional. Some researches had demonstrated MNs’ involvement in anti-stress responses in the yeast. Gene yfcC was recently found to modulate anaerobic survival of Escherichia. coli (E. coli) by us. In order to determine if disruption of yfcC could stimulate certain response manifesting by MNs, we developed a quick and simple method to quantify 20 nucleosides by using triple quadurupole liquid chromatography-tandem mass spectrometry (LC-MS/MS). Based on the consideration of the polar properties of the MNs, an Acquity UPLC HSS T3 column was adopted with the mobile phase of 10 mM ammonium acetate and acetonitrile at a flow-rate of 0.2 ml min-1. For the improvement of detection limit, multiple reaction monitoring mode as well as positive electrospray ionization was employed by using Triple Quadrupole mass spectrometry. The linearity of the detected MNs was excellent with R2 > 0.99 and the limits of detection were satisfied. The intra- and inter-day variation was ranged from 1.5% to 5.6% and 2.6% to 18.4%. Many of the included nuclesides could be readily quantified in E. coli and the most abundant of which was inosine. Additionally, distinct composition of the MNs could be distinguished between the wild type and genetic modified strains. This study indicated the effects of genetic manipulation on MNs in prokaryote also existed.To date, there are about 100 modified nucleosides (MNs) have been identified in RNA species and the majority of them were found in tRNAs. Except their common properties as components of RNAs, MNs have been confirmed to be of multifunctional. Some researches had demonstrated MNs’ involvement in anti-stress responses in the yeast. Gene yfcC was recently found to modulate anaerobic survival of Escherichia. coli (E. coli) by us. In order to determine if disruption of yfcC could stimulate certain response manifesting by MNs, we developed a quick and simple method to quantify 20 nucleosides by using triple quadurupole liquid chromatography-tandem mass spectrometry (LC-MS/MS). Based on the consideration of the polar properties of the MNs, an Acquity UPLC HSS T3 column was adopted with the mobile phase of 10 mM ammonium acetate and acetonitrile at a flow-rate of 0.2 ml min-1. For the improvement of detection limit, multiple reaction monitoring mode as well as positive electrospray ionization was employed by using Triple Quadrupole mass spectrometry. The linearity of the detected MNs was excellent with R2 > 0.99 and the limits of detection were satisfied. The intra- and inter-day variation was ranged from 1.5% to 5.6% and 2.6% to 18.4%. Many of the included nuclesides could be readily quantified in E. coli and the most abundant of which was inosine. Additionally, distinct composition of the MNs could be distinguished between the wild type and genetic modified strains. This study indicated the effects of genetic manipulation on MNs in prokaryote also existed