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Isolation protocols and mitochondrial content for plasma extracellular vesicles

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Abstract

Mitochondrial content has been reported outside of cells either within extracellular vesicles (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 isotope labeled internal standards to quantify 11 mitochondrial, 6 plasma membrane-specific, 4 endosomal membrane-specific, and 2 soluble proteins to evaluate the efficiency of chromatographic isolation of mitochondrial EVs. The isolation protocol includes 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 ultracentrifugation are contaminated with non-EV mitochondrial content that cannot be separated from EVs using size exclusion chromatography, but can be efficiently separated from EVs on the column with immobilized heparin.

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Acknowledgements

Certain commercial materials, instruments, and equipment are identified in this manuscript in order to specify the experimental procedure as completely as possible. In no case does such identification imply a recommendation or endorsement by the National Institute of Standards and Technology nor does it imply that the materials, instruments, or equipment identified are necessarily the best available for the purpose.

Funding

The research was supported by NIST budget.

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Authors and Affiliations

  1. Institute for Bioscience and Biotechnology Research, Rockville, MD, 20850, USA

    Ai Nguyen & Illarion V. Turko

  2. Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, MD, 20899, USA

    Illarion V. Turko

Authors
  1. Ai Nguyen
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  2. Illarion V. Turko
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Contributions

I.V. Turko designed the research, isolated EVs, and performed MRM experiments. A. Nguyen expressed and purified QconCATs. I.V. Turko and A. Nguyen analyzed results and wrote the paper.

Corresponding author

Correspondence to Illarion V. Turko.

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Source of biological material

Human plasma K2EDTA was purchased from BioreclamationIVT, Westbury, NY.

Conflict of interest

The authors declare no competing interests.

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Published in the topical collection Advances in Extracellular Vesicle Analysis with guest editors Lucile Alexandre, Jiashu Sun, Myriam Taverna, and Wenwan Zhong.

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Supplementary file1 (DOCX 31 KB)

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Nguyen, A., Turko, I.V. Isolation protocols and mitochondrial content for plasma extracellular vesicles. Anal Bioanal Chem 415, 1299–1304 (2023). https://doi.org/10.1007/s00216-022-04465-x

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  • DOI: https://doi.org/10.1007/s00216-022-04465-x

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