Protocol for cross-platform characterization of human and murine extracellular vesicles and particles

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Authors: Linda Bojmar ; Han Sang Kim ; Kei Sugiura ; Søren Heissel ; Serena Lucotti ; Michele Cioffi ; Kofi Ennu Johnson ; Leona Cohen-Gould ; Haiying Zhang ; Henrik Molina ; Irina R Matei ; David Lyden ; Ayuko Hoshino

MeSH: Animals ; Blotting, Western ; Computational Biology ; Extracellular Vesicles* ; Humans ; Mass Spectrometry ; Mice ; Proteomics*

Keywords: Cell Biology ; Cell Membrane ; Cell isolation ; Mass Spectrometry ; Model Organisms ; Molecular Biology ; Protein Biochemistry ; Proteomics

Abstract: Characterization of isolated extracellular vesicles and particles (EVPs) is crucial for determining functions and biomarker potential. Here, we present a protocol to analyze size, number, morphology, and EVP protein cargo and to validate EVP proteins in both humans and mice. We describe steps for nanoparticle tracking analysis, transmission electron microscopy, single-EVP immunodetection, EVP proteomic mass spectrometry and bioinformatic analysis, and EVP protein validation by ExoELISA and western blot analysis. This allows for EVP cross-validation across different platforms. For complete details on the use and execution of this protocol, please refer to Hoshino et al.1 © 2023 The Authors