The E. coli K12 strain was grown in standard LB medium, harvested, washed in PBS, and lysed in 4% SDS, 100 mm Tris, pH 8.5. Lysates were briefly boiled and DNA sheared using a Sonifier (Branson Model 250). Lysates were cleared by centrifugation at 15,000 × g for 15 min and precipitated with acetone. Proteins were resuspended in 8 m urea, 25 mm Tris, pH 8.5, 10 mm DTT. After 30 min of incubation, 20 mm iodoacetamide was added for alkylation. The sample was then diluted 1:3 with 50 mm ammonium bicarbonate buffer, and the protein concentration was estimated via tryptophan fluorescence emission assay. After 5 h of digestion with LysC (Wako Chemicals) at room temperature, the sample was further diluted 1:3 with ammonium bicarbonate buffer, and trypsin (Promega) digestion was performed overnight (protein-to-enzyme ratio of 60:1 in each case). E. coli peptides were then purified by using a C18 Sep Pak cartridge (Waters, Milford, MA) according to the manufacturer's instructions. UPS1 and UPS2 standards (Sigma-Aldrich) were resuspended in 30 μl of 8 m urea, 25 mm Tris, pH 8.5, 10 mm DTT and reduced, alkylated, and digested in an analogous manner, but with a lower protein-to-enzyme ratio (12:1 for UPS1 and 10:1 for UPS2, both LysC and trypsin). UPS peptides were then purified using C18 StageTips. E. coli and UPS peptides were quantified based on absorbance at 280 nm using a NanoDrop spectrophotometer (Fisher Scientific). For each run, 2 μg of E. coli peptides were then spiked with 0.15 μg of either UPS1 or UPS2 peptides, and about 1.6 μg of the mix was then analyzed via liquid chromatography combined with mass spectrometry on a Q Exactive (Thermo Fisher). Data were analyzed with MaxQuant as described above for the proteome dataset. All files were searched against the E. coli complete proteome sequences plus those of the UPS proteins and common contaminants.