The scope of the mass spectrometry approach of this study was to detect the presence or absence of bL19, bL 19-EGFP, bS20 and bS20-mCherry protein in 70S ribosome preparation from E. coli MC4100 or E. coli reporter strain (RN31). Therefore, in-gel digestion and nanoUPLC-ESI-Orbitrap-MS/MS were used.

Gel material corresponding to the E. coli MC4100 proteins bL19, bS20 and the E. coli reporter strain (RN31) proteins bL19-EGFP and bS20-mCherry were excised using an ExQuest spot cutter. After destaining and dehydration, gel pieces were rehydrated with trypsin solution and digested for 4 h at 37°C. Peptides were applied to a nanoACQUITY UPLC® precolumn (2G-VM Trap 5 µm Symmetry® C18 180 µm x 20 mm column) and separated on a nanoACQUITY UPLC® BEH C18 column (100 mm length, 75 µm internal diameter and 1.7 µm particle diameter, 35°C) at a flow rate of 0.3 μL/min using a gradient consisting of two linear slopes from 1 to 40% eluent B in 18.5 min and from 40 to 95% eluent B in 5.5 min. Eluent A was water containing 0.1% formic acid and eluent B was acetonitrile containing 0.1% formic acid. Samples were ionized using a PepSep emitter (Bruker Daltonics GmbH & Co. KG, Bremen, Germany) at a spray voltage of 1.8 kV. The temperature of the transfer capillary was set at 200 °C and the voltage of the tube lens was set at 100 V. A data dependent acquisition (DDA) approach created with Xcalibur software (version 2.0.7) was used: orbitrap resolution of 60,000 at m/z 400, precursor ion survey scans from m/z 400 to 2000, tandem mass spectra for the five most intense signals acquired in the linear ion trap (isolation width 2, activation Q 0.25, normalized collision energy 35%, activation time 30 ms) using dynamic exclusion for 60 s.

Gel bands obtained by SDS-PAGE separating the 70S ribosome of E. coli MC4100 and the 70S ribosome of the MC4100-based E. coli reporter strain (RN31).