Aminoglycoside antibiotics (AGAs) target the ribosome and induce mistranslation, yet which translation errors induce bacterial cell death is unclear. The analysis of cellular proteins by quantitative mass spectrometry shows that bactericidal AGAs induce not only single translation errors, but also clusters of errors in full-length proteins in vivo with as much as four amino acid substitutions in a row. The downstream errors in a cluster are much more frequent than the first error (0.4 vs. 10-3, respectively) and independent of the intracellular AGA concentration. The prevalence, length, and composition of error clusters depends not only on the misreading propensity of a given AGA, but also on its ability to inhibit ribosome translocation along the mRNA. Error clusters constitute a new class of misreading events in vivo that may constitute the predominant source of proteotoxic stress at low AGA concentration, which is particularly important for the autocatalytic uptake of the drugs.

Figures 1 and 3: DDA aquisition with quantification and identification runs. MaxQuant search results are manually validated in Skyline by MS1 filtering. Correct and missense peptides are quantified by LFQ. Figure 2, 4, and 5: Detection of error clusters by Parallel Reaction monitoring. Spike-in AQUA peptides are used for the identification of error clusters. Correct and missense peptides are quantified by LFQ. Raw data and search results used to build the spectrum libraries used in these analyses are available in the PRIDE repository with the ProteomeXchange ID PXD019188

Aminoglycoside induced misreading is quantified in the model protein EF-Tu.