Isolation of Bacterial Extracellular Vesicles and Identification of Their Protein Cargo

Bacterial membrane vesicles (BMVs) are important effectors in the pathogenesis, virulence, and biofilm formation during different bacterial infections. Because of their structure, BMVs can be applied as drug delivery systems (DDS) or in the production of immunogenic vaccines against different untreated diseases. In this sense, different antigens or immune stimulator molecules, such as proteins can be extracted for the development of such vaccines. Here, we describe a protocol adapted to be used in mycobacteria, Gram-positive, and Gram-negative bacteria for the isolation of BMVs, and further mass spectrometry-based characterization of their protein cargo.