Quantitative proteomic analysis of extracellular vesicle subgroups isolated by an optimized method combining polymer‐based precipitation and size exclusion chromatography

Journal of Extracellular Vesicles

卷:10

Karina Hernández‐Ortega¹  Ricardo Quiroz‐Baez²  Israel Pichardo‐Casas³  David A. Sinclair³  María del Pilar Ramos‐Godínez⁴  Osbaldo Resendis‐Antonio⁵  Eileen Uribe‐Querol⁶  Alfredo Hidalgo‐Miranda⁷  Juan A. Martínez‐Greene⁸  Eduardo Martínez‐Martínez⁸  Bogdan Budnik⁹ 

[1] Departamento de Biología Facultad de Química Universidad Nacional Autónoma de México Ciudad de México México;

[2] Departamento de Investigación Básica Instituto Nacional de Geriatría Mexico City Mexico;

[3] Department of Genetics Paul F. Glenn Labs for the Biology of Aging Harvard Medical School Boston Massachusetts USA;

[4] Electron Microscopy Laboratory Instituto Nacional de Cancerología Mexico City Mexico;

[5] Human Systems Biology Laboratory Instituto Nacional de Medicina Genómica Mexico City Mexico;

[6] Laboratorio de Biología del Desarrollo División de Estudios de Posgrado e Investigación Facultad de Odontología Universidad Nacional Autónoma de México Mexico City Mexico;

[7] Laboratorio de Genómica del Cáncer Instituto Nacional de Medicina Genómica Mexico City Mexico;

[8] Laboratory of Cell Communication & Extracellular Vesicles Instituto Nacional de Medicina Genómica Mexico City Mexico;

[9] Mass Spectrometry and Proteomics Resource Laboratory Division of Science Harvard University Cambridge Massachusetts USA;

关键词: breast cancer;    exosomes;    extracellular vesicles;    proteomics;    size‐exclusion chromatography;    ultracentrifugation;   

DOI  :  10.1002/jev2.12087

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown