Identification and Quantification of (t)RNA Modifications in Pseudomonas aeruginosa by Liquid Chromatography–Tandem Mass Spectrometry

Transfer RNA (tRNA) modifications impact the structure and function of tRNAs, thus affecting the efficiency and fidelity of translation. In the opportunistic pathogen Pseudomonas aeruginosa translational regulation plays an important but less defined role in adaptation to changing environments. In this study, we have explored tRNA modifications in P. aeruginosa through LC‐MS/MS approaches. Neutral loss scanning (NLS) demonstrated the potential to identify previously unknown modifications, whereas multiple reaction monitoring (MRM) was able to detect modifications with high specificity and sensitivity. In this study, the MRM‐based external calibration method allowed for quantification of the four canonical and 32 modified ribonucleosides, out of which 21 tRNA modifications were quantified in the total tRNA pool of P. aeruginosa PA14. We also purified the single tRNA isoacceptors tRNA‐ArgUCU, tRNA‐LeuCAA, and tRNA‐TrpCCA and determined their specific modification patterns, both qualitatively and quantitatively. Deeper insights into the nature and dynamics of tRNA modifications in P. aeruginosa should pave the way for further studies on post‐transcriptional gene regulation as a relatively unexplored molecular mechanism of controlling bacterial pathogenicity and mode of growth. Controlling bacterial behavior: We have explored (t)RNA modifications in P. aeruginosa by LC‐MS/MS methods, both qualitatively and quantitatively. This analytical biological approach has shed light on the dynamic landscape of tRNA modifications. Enzymes that modulate the modification status of selected tRNAs could represent dynamic regulators of gene expression with an impact on protein levels.