Ziptip filter

Protein extraction and sample preparation for LC–MS/MS measurements were performed according to previously published protocols^{32 (link)}. In brief, the proteins were prepared by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) for sample decontamination and in-gel digested with 0.5 µg trypsin (Sigma-Aldrich, St. Louis, USA), overnight. The extracted peptides were desalted using ZipTip filter (Thermo Fisher Scientific) following the manufacturer’s instructions and analyzed using liquid chromatography (HPLC, Ultimate 3000 RSLCnano, Dionex/Thermo Fisher Scientific, Idstein, Germany) coupled via a TriVersa NanoMate (Advion, Ltd., Harlow, UK) source in LC chip coupling mode with a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). The samples were measured according to the settings outlined in Starke et al.^{33 (link)}. The acquired raw data were searched by Sequest HT in Proteome Discoverer v2.1 (Thermo Fisher Scientific) against an in-silico protein database containing all bacteria (> 10⁶ sequences), downloaded 2017 from Uniprot. We considered only proteins with a false-discovery rate of < 1%.

For sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the protein pellet was resuspended with 20 µL SDS loading buffer and incubated for 5 min in a Thermomixer at 95 °C and 1400 rpm. After SDS-PAGE and staining with colloidal Coomassie brilliant blue (Merck, Darmstadt, Germany) overnight, the colored gel bands containing all proteins were cut out and were sliced into smaller gel pieces. Then, the gel bands were destained by two rinses with H₂O for 30 min at room temperature. Proteins in each band were modified with 10 mM Dithioerythritol (DTT) and 100 mM 2-iodacetamide (IAA) and incubated for 30 min at room temperature. We applied 20 µg alkylated proteins which were proteolytically digested using 0.5 µg trypsin (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C, overnight. Digestion was stopped by adding 10 mM ammonium bicarbonate in 0.1% formic acid (FA). After peptide extraction using extraction buffer (50% acetonitrile and 5% formic acid), the samples were evaporated using a SpeedVac for 2 h and stored at −20 °C. The extracted peptides were desalted using ZipTip filter (Thermo Fischer Scientific, Waltham, MA, USA) following the manufacturer’s instructions. Peptides were dissolved in 0.1% FA and injected into the liquid chromatography–mass spectrometer.