title: Isolation and identification of membrane vesicle-associated proteins in Gram-positive bacteria and mycobacteria.
abstract: 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.
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Rafael
authors.initials: R
authors.lastname: Prados-Rosales
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Lisa
authors.initials: L
authors.lastname: Brown
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Arturo
authors.initials: A
authors.lastname: Casadevall
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA ; Department of Analytical Chemistry, Complutense University of Madrid, Madrid 28040, Spain.
authors.forename: Sandra
authors.initials: S
authors.lastname: Montalvo-Quirós
authors.affiliation: Department of Analytical Chemistry, Complutense University of Madrid, Madrid 28040, Spain.
authors.forename: Jose L
authors.initials: JL
authors.lastname: Luque-Garcia
keywords: Gram positive bacteria; Membrane vesicles; Protein content; Vesicles isolation