1. Analysis of Pseudomonas aeruginosa Cell Envelope Proteome by Capture of Surface-Exposed Proteins on Activated Magnetic Nanoparticles
- Author
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Pierluigi Mauri, Alessandra Bragonzi, M. Miriani, Mauro Marengo, Davide Vecchietti, Lara Cova, Dario Di Silvestre, Giovanni Bertoni, Eleonora Franceschi, and Francesco Bonomi
- Subjects
Proteomics ,Proteases ,Proteome ,lcsh:Medicine ,Plasma protein binding ,Biology ,DNA-binding protein ,Biochemistry ,Microbiology ,Cell membrane ,03 medical and health sciences ,0302 clinical medicine ,Model Organisms ,Bacterial Proteins ,Microbial Physiology ,medicine ,Gram Negative ,Bacterial Physiology ,lcsh:Science ,Magnetite Nanoparticles ,Microbial Pathogens ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,Spectrometric Identification of Proteins ,Proteomic Databases ,lcsh:R ,Cell Membrane ,Proteins ,Reproducibility of Results ,Bacteriology ,Bacterial Pathogens ,Transmembrane Proteins ,medicine.anatomical_structure ,Membrane protein ,Cytoplasm ,030220 oncology & carcinogenesis ,Pseudomonas aeruginosa ,Prokaryotic Models ,lcsh:Q ,Cell envelope ,Research Article ,Protein Binding - Abstract
We report on specific magneto-capturing followed by Multidimensional Protein Identification Technology (MudPIT) for the analysis of surface-exposed proteins of intact cells of the bacterial opportunistic pathogen Pseudomonas aeruginosa. The magneto-separation of cell envelope fragments from the soluble cytoplasmic fraction allowed the MudPIT identification of the captured and neighboring proteins. Remarkably, we identified 63 proteins captured directly by nanoparticles and 67 proteins embedded in the cell envelope fragments. For a high number of proteins, our analysis strongly indicates either surface exposure or localization in an envelope district. The localization of most identified proteins was only predicted or totally unknown. This novel approach greatly improves the sensitivity and specificity of the previous methods, such as surface shaving with proteases that was also tested on P. aeruginosa. The magneto-capture procedure is simple, safe, and rapid, and appears to be well-suited for envelope studies in highly pathogenic bacteria. © 2012 Vecchietti et al.
- Published
- 2012