2.2. Protein extraction and digestion

Pellets from P. aeruginosa PAO1 cells under the different conditions were washed and resuspended in 1 ml of PBS supplemented with 1/1000 Protease Inhibitor Cocktail (Complete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets). PAO1 biofilm was washed with PBS and cells were collected to protein extraction. Cells from the different samples were lysed by sonication (Labsonic U) for 3 times, 1 min each, and centrifuged for 20 min at 6000g at 4°C. Cell debris was removed by centrifugation (14000g, 10 min, 4°C). Proteins were frozen at −80ºC until used. Protein concentration was measured using Bradford assay (Biorad).

The secretome from the T3SS-induction sample was collected by centrifugation at 7000g, 10 min, 4°C and supernatants were filtered through a 0.2 μm pore size Nalgene Disposable Filter Unit (Thermo Fisher Scientific) to remove the remaining cells. Proteins were precipitated with methanol/chloroform. The pellet was washed 2 times with PBS and resuspended in 0.5 M Triethylammonium bicarbonate (TEAB) supplemented with 1/1000 Protease Inhibitor Cocktail.

In the case of the inner and outer membranes enriched fractions, cells were pelleted and washed with PBS and resuspended in 1.5 ml of 20 mM HEPES pH 8.0. Then, lysis was reached by sonication for 3 times, 1 min each, and centrifuged for 20 min at 6000g at 4°C. After this, supernatant was collected and ultracentrifuged at 100000g during 1h at 4°C. Then, supernatant was collected and pellet was resuspended in 1.5 ml 20 mM HEPES pH 8.0 and the suspension was ultracentrifuged at 100000 g during 30 at 4°C. Total membranes were pelleted and resuspended in 1.5 ml of 20 mM HEPES pH 8.0 + 15 μl TRITON X-100, ultracentrifuged at 100000g during 1h at 4ºC. Outer membranes were located in the pellet and inner membranes were enriched in the supernatant. Inner membranes were collected. The pellet was washed in 1.5 ml of 20 mM HEPES pH 8.0, ultracentrifuged at 100000g during 1h at 4°C and pellet, enriched in outer membranes, was resuspended in 300 μl of 20 mM HEPES pH 8.0.

One hundred μg of the different PAO1 cytoplasmic extracts were loaded onto conventional SDS-PAGE 10% Bis-Tris gels (mini-protean TGX Stain-free precast Gels, BioRad). The gel was stained with Coomassie blue and each lane was cut into 10 bands. Gel slices were cut into 1 mm³ cubes, washed twice with water and dehydrated with 100% ACN (v/v).

One hundred μg of protein of subproteomes (outer and inner membranes and T3SS secretome) were loaded in a conventional SDS-PAGE gel (1mm-thick, 4% stacking, and 12% resolving). Then run was stopped as soon as the front entered into the resolving gel, so that the whole proteome became concentrated in the stacking/resolving gel interface. The unseparated protein bands were visualized by Coomassie staining, excised, cut into cubes (1mm³), washed twice with water and dehydrated with 100% ACN (v/v). All the gel slices were incubated with 10 mM DTT in 50 mM NH₄HCO₃ for 30 min at 56°C for protein reduction; alkylation was carried out with 50 mM IAA in 50 mM ammonium bicarbonate solution.

The gel pieces were washed with 50% ACN (v/v), and then washed again with 10 mM NH₄HCO₃, dehydrated with 100% ACN (v/v), and then dried in a vacuum concentrator. The gel pieces were rehydrated by adding sequence grade-modified trypsin (Roche) 1:20 in 50mM NH₄HCO₃ and incubated overnight at room temperature in the dark for protein digestion. Supernatants were transferred to clean tubes, and gel pieces were incubated in 50mM NH₄HCO₃ at 50 °C for 1 h. Then, remaining peptides were collected by incubation with 5% formic acid for 15 min and with 100% ACN for 15 min more. The extracts were combined, and the organic solvent was removed in a vacuum concentrator.

The tryptic eluted peptides were dried by speed-vacuum centrifugation and then desalted onto StageTip C18 Pipette tips (Thermo Scientific) until the mass spectrometric analysis. The 10 slices of the whole proteomes were combined and 5 fractions were used for the mass spectrometry analysis.