Quantitative Proteomics Reveals Antibiotics Resistance Function of Outer Membrane Proteins in Aeromonas hydrophila

ABSTRACT: Each colony of A. hydrophila and the OXY-R strain were incubated in 5 mL LB medium overnight, and then diluted in 100 mL fresh LB medium at a ratio of 1:100 and subsequently cultured until the optical density at 600 nm (OD600) reached 1.0. The cultures were harvested via centrifuging for 20 min at 10,000 x g, 4°C, and then the bacterial cells were washed with cold phosphate buffered saline (PBS, pH 7.4) for three times. The cell pellets were resuspending in the 10 mL ultrasonic buffer (50 mM Tris-HCl, pH 7.4, 1 mM PMSF) and disrupted with intermittent sonic oscillation for a total of 30 minutes at 9 seconds intervals on ice. Subsequently, the cell debris and unbroken cells were separated by centrifugation at 8,000 x g for 20 min at 4°C. Then the supernatant was centrifuged at 100,000 x g for 1 h at 4°C in the Optima LE-80 K Ultracentrifuge (Beckman, Palo Alto, CA, USA). After the pellet was dissolved with 2% sodium lauroyl sarcosine (in 50 mM Tris-HCl, pH 7.5) for 40 min at room temperature (RT), the pellets were ultracentrifuged again at 100,000 x g for 1 h at 4°C. Finally, the precipitate was dissolved in an appropriate volume of SDT buffer (4 % SDS, 0.1 M DTT [dithiothreitol], and 0.5 M triethylammonium bicarbonate buffer [TEAB, pH 8.5]). The protein concentration was determined using the Bradford method and then stored at -20°C until subsequent use.The proteins were digested with trypsin after being reduced with DTT and alkylated with Iodoacetamide using a filter-aided sample preparation method (Tanca et al., 2013; Li et al., 2016b). After washing three times with 0.5 M TEAB followed by fractionation using the 10 kDa ultrafiltration system (Millipore, Billerica, MA, USA), about 100 μg digested peptide from each group, including two biological replicates, was taken out for further labeled using TMT isobaric and isotopic mass-tagging kits (Thermo Fisher Scientific, MA, USA), which was performed according to instructions from the kit.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Aeromonas Hydrophila Subsp. Hydrophila Atcc 7966

SUBMITTER: Xiangmin Lin  

LAB HEAD: Xiangmin Lin

PROVIDER: PXD009622 | Pride | 2018-11-23

REPOSITORIES: Pride