We used a combination of genetic and proteomic approaches to characterize tmRNA (ssrA) activity in the genome-reduced bacterium Mycoplasma pneumoniae. For this, we generated tmRNA mutants encoding a tag resistant to proteolysis. Endogenous protein tagging by the mutant tmRNA gene (ssrAmk) was then examined by immunoprecipitation (IP) enrichment followed by LC-MS/MS analysis.