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.

Analysis of Pseudomonas aeruginosa Cell Envelope Proteome by Capture of Surface-Exposed Proteins on Activated Magnetic Nanoparticles / D. Vecchietti, D. Di Silvestre, M. Miriani, F. Bonomi, M. Marengo, A. Bragonzi, L. Cova, E. Franceschi, P. Mauri, G. Bertoni. - In: PLOS ONE. - ISSN 1932-6203. - 7:11(2012), p. e51062.e51062.

Analysis of Pseudomonas aeruginosa Cell Envelope Proteome by Capture of Surface-Exposed Proteins on Activated Magnetic Nanoparticles

D. Vecchietti;M. Miriani;F. Bonomi;M. Marengo;G. Bertoni
2012

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.
Settore BIO/10 - Biochimica
Settore BIO/19 - Microbiologia Generale
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/215079
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