Open AccessQuantitative proteomic analysis of extracellular vesicle subgroups isolated by an optimized method combining polymer‐based precipitation and size exclusion chromatography
Author(s) -
MartínezGreene Juan A.,
HernándezOrtega Karina,
QuirozBaez Ricardo,
ResendisAntonio Osbaldo,
PichardoCasas Israel,
Sinclair David A.,
Budnik Bogdan,
HidalgoMiranda Alfredo,
UribeQuerol Eileen,
RamosGodínez María del Pilar,
MartínezMartínez Eduardo
Publication year - 2021
Publication title -
journal of extracellular vesicles
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.94
H-Index - 68
ISSN - 2001-3078
DOI - 10.1002/jev2.12087
Subject(s) - nanoparticle tracking analysis , extracellular vesicles , microvesicles , size exclusion chromatography , western blot , chemistry , cd81 , extracellular vesicle , vesicle , protein purification , chromatography , computational biology , biology , microbiology and biotechnology , biochemistry , membrane , immunology , microrna , hepatitis c virus , virus , gene , enzyme
Abstract The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in their composition at a cellular and tissue level. Current isolation methods fail to efficiently separate EV subtypes for proteomic and functional analysis. The aim of this study was to develop a reproducible and scalable isolation workflow to increase the yield and purity of EV preparations. Through a combination of polymer‐based precipitation and size exclusion chromatography (Pre‐SEC), we analyzed two subsets of EVs based on their CD9, CD63 and CD81 content and elution time. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blot assays. To evaluate differences in protein composition between the early‐ and late‐eluting EV fractions, we performed a quantitative proteomic analysis of MDA‐MB‐468‐derived EVs. We identified 286 exclusive proteins in early‐eluting fractions and 148 proteins with a differential concentration between early‐ and late‐eluting fractions. A density gradient analysis further revealed EV heterogeneity within each analyzed subgroup. Through a systems biology approach, we found significant interactions among proteins contained in the EVs which suggest the existence of functional clusters related to specific biological processes. The workflow presented here allows the study of EV subtypes within a single cell type and contributes to standardizing the EV isolation for functional studies.