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Isolation protocols and mitochondrial content for plasma extracellular vesicles



Ai Nguyen, Illarion Turko


Mitochondrial content has been reported outside of cells either within extracellular ves-icles (EVs) or as free mitochondria. Mitochondrial EVs can potentially play multiple physiological and pathophysiological roles. To understand their functions, isolation protocols to separate mitochondrial EVs from other mitochondrial content need to be established. In the present work, we use a multiple reaction monitoring assay with iso-tope labeled internal standards to quantify 11 mitochondrial, 6 plasma membrane-specific, 4 endosomal membrane-specific and 2 soluble proteins to evaluate the effi-ciency of chromatographic isolation of mitochondrial EVs. The isolation protocol in-cludes ultracentrifugation, size exclusion chromatography and chromatography on immobilized heparin. All protein concentrations were normalized to the concentration of ATP synthase alpha subunit to generate a ratio that allows comparison of different samples obtained during the isolation. We have shown that initial samples after ultra-centrifugation are contaminated with non-EVs mitochondrial content that cannot be separated from EVs using size exclusion chromatography, but can be efficiently sepa-rated from EVs on the column with immobilized heparin.
Analytical and Bioanalytical Chemistry


Mass spectrometry, Targeted Proteomics, Extracellular vesicles, Mitochondria, Heparin-Sepharose


Nguyen, A. and Turko, I. (2022), Isolation protocols and mitochondrial content for plasma extracellular vesicles, Analytical and Bioanalytical Chemistry, [online], (Accessed May 25, 2024)


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Created December 2, 2022, Updated February 24, 2023