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Isolation and identification of membrane vesicle-associated proteins in Gram-positive bacteria and mycobacteria

Many intracellular bacterial pathogens naturally release membrane vesicles (MVs) under a variety of growth environments. For pathogenic bacteria there are strong evidences that released MVs are a delivery mechanism for the release of immunologically active molecules that contribute to virulence. Ide...

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Autores principales: Prados-Rosales, Rafael, Brown, Lisa, Casadevall, Arturo, Montalvo-Quirós, Sandra, Luque-Garcia, Jose L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4472924/
https://www.ncbi.nlm.nih.gov/pubmed/26150943
http://dx.doi.org/10.1016/j.mex.2014.08.001
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author Prados-Rosales, Rafael
Brown, Lisa
Casadevall, Arturo
Montalvo-Quirós, Sandra
Luque-Garcia, Jose L.
author_facet Prados-Rosales, Rafael
Brown, Lisa
Casadevall, Arturo
Montalvo-Quirós, Sandra
Luque-Garcia, Jose L.
author_sort Prados-Rosales, Rafael
collection PubMed
description Many intracellular bacterial pathogens naturally release membrane vesicles (MVs) under a variety of growth environments. For pathogenic bacteria there are strong evidences that released MVs are a delivery mechanism for the release of immunologically active molecules that contribute to virulence. Identification of membrane vesicle-associated proteins that can act as immunological modulators is crucial for opening up new horizons for understanding the pathogenesis of certain bacteria and for developing novel vaccines. In this protocol, we provide all the details for isolating MVs secreted by either mycobacteria or Gram-positive bacteria and for the subsequent identification of the protein content of the MVs by mass spectrometry. The protocol is adapted from Gram-negative bacteria and involves four main steps: (1) isolation of MVs from the culture media; (2) purification of MVs by density gradient ultrucentrifugation; (3) acetone precipitation of the MVs protein content and in-solution trypsin digestion and (4) mass spectrometry analysis of the generated peptides and protein identification. Our modifications are: • Growing Mycobacteria in a chemically defined media to reduce the number of unrelated bacterial components in the supernatant. • The use of an ultrafiltration system, which allows concentrating larger volumes. • In solution digestion of proteins followed by peptides purification by ziptip.
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spelling pubmed-44729242015-07-06 Isolation and identification of membrane vesicle-associated proteins in Gram-positive bacteria and mycobacteria Prados-Rosales, Rafael Brown, Lisa Casadevall, Arturo Montalvo-Quirós, Sandra Luque-Garcia, Jose L. MethodsX Article Many intracellular bacterial pathogens naturally release membrane vesicles (MVs) under a variety of growth environments. For pathogenic bacteria there are strong evidences that released MVs are a delivery mechanism for the release of immunologically active molecules that contribute to virulence. Identification of membrane vesicle-associated proteins that can act as immunological modulators is crucial for opening up new horizons for understanding the pathogenesis of certain bacteria and for developing novel vaccines. In this protocol, we provide all the details for isolating MVs secreted by either mycobacteria or Gram-positive bacteria and for the subsequent identification of the protein content of the MVs by mass spectrometry. The protocol is adapted from Gram-negative bacteria and involves four main steps: (1) isolation of MVs from the culture media; (2) purification of MVs by density gradient ultrucentrifugation; (3) acetone precipitation of the MVs protein content and in-solution trypsin digestion and (4) mass spectrometry analysis of the generated peptides and protein identification. Our modifications are: • Growing Mycobacteria in a chemically defined media to reduce the number of unrelated bacterial components in the supernatant. • The use of an ultrafiltration system, which allows concentrating larger volumes. • In solution digestion of proteins followed by peptides purification by ziptip. Elsevier 2014-08-19 /pmc/articles/PMC4472924/ /pubmed/26150943 http://dx.doi.org/10.1016/j.mex.2014.08.001 Text en © 2014 The Authors http://creativecommons.org/licenses/by/3.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).
spellingShingle Article
Prados-Rosales, Rafael
Brown, Lisa
Casadevall, Arturo
Montalvo-Quirós, Sandra
Luque-Garcia, Jose L.
Isolation and identification of membrane vesicle-associated proteins in Gram-positive bacteria and mycobacteria
title Isolation and identification of membrane vesicle-associated proteins in Gram-positive bacteria and mycobacteria
title_full Isolation and identification of membrane vesicle-associated proteins in Gram-positive bacteria and mycobacteria
title_fullStr Isolation and identification of membrane vesicle-associated proteins in Gram-positive bacteria and mycobacteria
title_full_unstemmed Isolation and identification of membrane vesicle-associated proteins in Gram-positive bacteria and mycobacteria
title_short Isolation and identification of membrane vesicle-associated proteins in Gram-positive bacteria and mycobacteria
title_sort isolation and identification of membrane vesicle-associated proteins in gram-positive bacteria and mycobacteria
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4472924/
https://www.ncbi.nlm.nih.gov/pubmed/26150943
http://dx.doi.org/10.1016/j.mex.2014.08.001
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