Computational protocol: Outer-Inner Membrane Vesicles Naturally Secreted by Gram-Negative Pathogenic Bacteria

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Protocol publication

[…] A proteomic analysis of purified MVs from N. gonorrhoeae was carried out using one-dimensional (1-D) SDS-PAGE and nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis as described by Pérez-Cruz et al. with some modifications []. Briefly, proteins were in-gel digested with trypsin, and the tryptic peptides were extracted from the gel matrix, pooled, and dried in a vacuum centrifuge. The dried-down peptide mixture was analyzed in a nanoAcquity liquid chromatographer (Waters) coupled to an LTQ-Orbitrap Velos (Thermo Scientific) mass spectrometer. A database was created merging all Uniprot-SwissProt and Uniprot-TrEMBL entries for Neisseria gonorrhoeae with Uniprot-Swissprot all (September 2013). The software Thermo Proteome Discover (v.1.3.0.339) was used to perform a search by the Sequest search engine against this database. The results were filtered and only proteins identified with at least 2 high confidence peptides (FDR≤ 0.01) were included. Bacterial protein subcellular localization was predicted with the software PSORTb v3.0.2 []. […]

Pipeline specifications

Software tools Comet, PSORTb
Applications MS-based untargeted proteomics, Protein sequence analysis
Organisms Bacteria, Neisseria gonorrhoeae, Pseudomonas aeruginosa PAO1, Acinetobacter baumannii