a EF-Tu-derived peptides with one (gray) or two (red) errors in Str-treated wt cells and in untreated ram cells. Shown are means of three technical replicates (n = 3). b Error clusters induced by Str are absent in the ram strain. Str concentration (4 µM) was chosen to achieve the same Ef in wt and ram cells. Peptides with error clusters were detected by QRAS and SRM (rdotp = 1, fragment ions color coded). c Enrichment of error clusters in aggregates. After treating cells with Str (8 µM), EF-Tu was isolated from the soluble cell fraction and the insoluble aggregates and peptides analyzed by DDA. Shown are the means of three technical replicates (n = 3). d Time course (left panel) and concentration dependence (right panel) of Str treatment for D208E-F211L error cluster detected by PRM. Shown are means ± SD of three technical replicates (n = 3). e Str-induced error clusters with three and four substitutions. Target peptides were enriched by QRAS and detected by PRM. f Examples of PRM validation for error clusters in different proteins induced by Str (8 µM). g Types of misreading events in error clusters. Errors with ambiguous classification of the misreading position are shown in gray; in cases where misreading can arise from mismatches at different positions, the errors were classified as originating from the 3rd > 1st > 2nd position mismatch, according to their response to AGA treatment9–11. h Effect of different AGAs. The error cluster D208E-F211L in EF-Tu detected by PRM upon treatment with spectinomycin (Spc), streptomycin (Str), dihydrostreptomycin (DHS), paromomycin (Par), apramycin (Apr), sisomycin (Sis), gentamycin (Gen), geneticin (G418), hygromycinB (HygB), neomycin (Neo), tobramycin (Tob), kanamycin A (KanA), kanamycin B (KanB), amikacin (Amk), or neamine (Nea) at concentrations that gave rise to maximum misreading (‘Methods’). Shown are means ± SD of three technical replicates (n = 3). See also Supplementary Figs. 1–3 and Source data file.