Flexanalysis software v 3

For peptide identification, LIFT-TOF/TOF spectra were acquired using the UltrafleXtreme™ MALDI-TOF/TOF mass spectrometer without additional collision gas. Analyses were performed using appropriate acquisition settings as previously reported [11] . MS/MS data were processed using FlexAnalysis™ software v. 3.3 (Bruker Daltonics, Germany). Database searching was performed by an in-house Mascot search engine (Version: 2.4.1) with the same parameters already described [11] .

All MALDI-TOF experiments were performed at CAARU, Sultan Qaboos University, on UltraFlextreme (Bruker Daltonics, Bremen, Germany) operating in positive reflectron mode in the m/z range of 50–2000 Da. Stainless steel MTP 384 target plate was used for all the molecular weight analysis. Dihydroxy benzoic acid (DHB) dried droplet protocol described in Bruker manual was employed for sample preparation and spotting. Two micro liter of 2, 5-DHB matrix (20 mg/ml) in TA 30 (30:70 v/v ACN:TFA 0.1%TFA) was premixed with 2 μl of the sample solution. One micro liter of the mixture was applied to the ground steel target plate, dried at room temperature. The spectra were acquired using FlexControl software v3.3 (Bruker Daltonics, Bremen, Germany). A SmartBeam-II laser, set at a frequency of 1000 Hz, was used for ionization. The laser strength was optimized at 25–35%. A summed spectrum was obtained for each MALDI-spot. Peaks were detected using the SNAP peak detection algorithm and a baseline subtraction was carried out using “TopHat” algorithm. The MALDI-TOF spectra were externally calibrated using a commercially available peptide mix (peptide calibration standard II, Part-No #222570, Bruker Daltonics, Germany). FlexAnalysis Software v3.3 (Bruker Daltonics) was used for visualization and initial data processing.

The bacterial suspensions were set to 0.5 McFarland standard turbidity in deionized water, pelleted (13,800 × g for 5 min at room temperature [rt]), and resuspended in 300 μL of deionized water. Then, 900 μL of 99.8% (vol/vol) ethanol (Carl Roth, Karlsruhe, Germany) was added. Following two centrifugation steps (9,600 × g for 2 min at rt) and air drying (4 to 6 min), the pellets were resuspended in 20 μL of 70% (vol/vol) formic acid (Thermo Fisher Scientific, Waltham, MA, USA). The suspensions were mixed with 20 μL of acetonitrile (Carl Roth, Karlsruhe, Germany) and centrifuged (9,600 × g for 2 min at rt). Next, 1 μL of supernatants was spotted on a MALDI target plate (MBT Biotarget 96, Bruker Daltonik, Bremen, Germany) and left to dry at rt, and 1 μL of 130 μM 2,5-dihydroxybenzoic acid matrix (Sigma-Aldrich, St. Louis, MO, USA) was added onto each protein-containing spot. The extracted membrane proteins were analyzed by MALDI-TOF MS using a Microflex smart instrument (mass range of 2 to 40 kDa, laser intensity of 70%, number and frequency of shots of 200; Bruker Daltonik, Bremen, Germany). The analysis of spectra was performed using flexAnalysis software v.3.3 (Bruker Daltonik, Bremen, Germany).

The bacterial suspensions were set to 0.5 McFarland standard turbidity in deionized water, pelleted (13,800 × g for 5 min at room temperature [rt]), and resuspended in 300 μL of deionized water. Then, 900 μL of 99.8% (vol/vol) ethanol (Carl Roth, Karlsruhe, Germany) was added. Following two centrifugation steps (9,600 × g for 2 min at rt) and air drying (4 to 6 min), the pellets were resuspended in 20 μL of 70% (vol/vol) formic acid (Thermo Fisher Scientific, Waltham, MA, USA). The suspensions were mixed with 20 μL of acetonitrile (Carl Roth, Karlsruhe, Germany) and centrifuged (9,600 × g for 2 min at rt). Next, 1 μL of supernatants was spotted on a MALDI target plate (MBT Biotarget 96, Bruker Daltonik, Bremen, Germany) and left to dry at rt, and 1 μL of 130 μM 2,5-dihydroxybenzoic acid matrix (Sigma-Aldrich, St. Louis, MO, USA) was added onto each protein-containing spot. The extracted membrane proteins were analyzed by MALDI-TOF MS using a Microflex smart instrument (mass range of 2 to 40 kDa, laser intensity of 70%, number and frequency of shots of 200; Bruker Daltonik, Bremen, Germany). The analysis of spectra was performed using flexAnalysis software v.3.3 (Bruker Daltonik, Bremen, Germany).