Enterobacter cloacae OMVs
Description
Enterobacter cloacae OMVs. Data include three biological replicates for the OMVs generated at the stationary phase of the growth of Enterobacter cloacae.
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The experimental steps were described in https://doi.org/10.1016/j.jprot.2020.103994. OMV (outer membrane vesicle) samples were lysed using 1% Triton X-100 and concentrated using TCA protein precipitation. The protein pellet was resuspended and homogenized in 1.5× Bolt LDS sample buffer and Bol sample reducing agent and heated at 70 °C for 10 mins. The samples were run 1/3rd of the length through 4–12% Bis-Tris SDS PAGE gel and stained using GelCode Blue . The sample lanes in the gel are cut into 1–2 mm3 pieces, washed overnight with 50% methanol and 5% acetic acid, alkylated with 50 mM iodoacetamide, and reduced by adding 10 mM DTT. Proteins were digested overnight in 50 mM ammonium bicarbonate with trypsin at 37 °C. Following day, the proteins were extracted with acetonitrile and formic acid. The isolated peptides were lyophilized in a vacuum centrifuge and stored at −20 °C.The peptide samples were analyzed by Ultrahigh-Performance Liquid Chromatography (UHPLC) coupled to Orbitrap Fusion mass spectrometer (Thermo Scientific). The data analysis was done using Proteome Discoverer. he MS/MS spectra were further analyzed using Mascot (Matrix Science, London, UK; version 2.7.0.1). The mascot was set up to search the UniProt Enterobacter_cloacae database, where the digestion enzyme was trypsin. The mascot-based search was set up to include mass tolerance of 10.00 PPM for the parent ions and 1.00 Da for the fragment ions. The following protein modifications have been included in the search: O + 18 of pyrrolysine, as well as carbamidomethyl of cysteine, were specified as fixed modifications. Moreover, Gln- > pyro-Glu of the N-terminus, deamidation of asparagine and glutamine and oxidation of methionine were specified as variable modifications of the proteins. Scaffold (version 4.11.0, Proteome Software Inc., Portland, OR) was used as a tool to validate MS/MS-based peptide and protein identifications further, where the protein probabilities were established by protein Prophet algorithm [48]. Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least two identified peptides. Protein grouping has been used, where proteins that contain similar peptides, and thus cannot be differentiated based on the fragmentation-based MS/MS analysis, were grouped into clusters, and the top hits were recorded, where appropriate. The calculated protein FDR for the presented dataset is 0.1%, and peptide FDR is 0.03%, based on the decoy databank search performed on the same dataset.