TY - JOUR
T1 - Isolation and identification of membrane vesicle-associated proteins in Gram-positive bacteria and mycobacteria
AU - Prados-Rosales, Rafael
AU - Brown, Lisa
AU - Casadevall, Arturo
AU - Montalvo-Quirós, Sandra
AU - Luque-Garcia, Jose L.
N1 - Publisher Copyright:
© 2014 The Authors.
PY - 2014
Y1 - 2014
N2 - 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. MethodProtein content of bacteria membrane vesicles.
AB - 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. MethodProtein content of bacteria membrane vesicles.
KW - Gram positive bacteria
KW - Membrane vesicles
KW - Protein content
KW - Vesicles isolation
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U2 - 10.1016/j.mex.2014.08.001
DO - 10.1016/j.mex.2014.08.001
M3 - Article
C2 - 26150943
AN - SCOPUS:84907537129
SN - 2215-0161
VL - 1
SP - e124-e129
JO - MethodsX
JF - MethodsX
ER -