Proteinpilot

Isolated SDS-PAGE gel plugs were subjected to proteolysis by proteome grade trypsin (Sigma Aldrich, St. Louis, MO, USA) at pH 8, 37 °C, overnight before analysis by mass spectrometry. The peptide mix was desalted using C18 ZipTips (Millipore, Burlington, MA, USA) and 1 µL of this solution was combined with 1 µL of a 3 mg⁻¹mL α-cyano-4-hydroxycinnamic acid (60% acetonitrile, 1 mM ammonium diphosphate) and spotted onto MALDI targets. All MALDI-MS experiments were performed using a 5800 MALDI-TOF/TOF (Applied Biosystems, Foster City, CA, USA). The MS data were acquired using the reflectron detector in positive mode (700–4500 Da, 1900 Da focus mass) using 300 laser shots (50 shots per sub-spectrum). Collision-induced dissociation tandem MS spectra were acquired using 1 kV of collision energy. All MS and MS/MS data were searched against the UniProt protein sequence database using Protein Pilot (Applied Biosystems, Foster City, CA, USA) software. The database search parameters used for Mascot (Matrix Science, London, UK) were the following: mass tolerance: 0.5 Da, taxonomy: phage and E. coli, enzyme: trypsin, missed cleavages: 1, and variable modifications: oxidation (M), deamidation (N, Q).

Proteomic characterization of LDEVs was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS, nano-LC from LC Packings/Dionex, and Qstar XL from Applied Biosystems). LDEV samples were suspended in Novex® (Thermo Scientific) reducing sample buffer and heated for 10 min at 70 °C. Samples were run on Novex® 4-20% Tris-Glycine gradient gels and stained with SimplyBlue® SafeStain (Life Technologies) for 1 h followed by destaining with water. Gel bands were excised and digested with modified Trypsin (Promega). Tryptic digests were fractionated with a reversed-phase column and the column eluate introduced onto a Qstar XL mass spectrometer via ESI. Protein identifications were performed with ProteinPilot (Applied Biosystems) using the L. plantarum WCFS1 reference sequence database from UniProt and NCBI. To increase confidence, a further manual inspection was carried out to select the proteins associated with at least two unique as the potential candidates [44 (link)–46 (link)].