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Analysis of Extracellular Vesicle and Contaminant Markers in Blood Derivatives Using Multiple Reaction Monitoring

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Serum/Plasma Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2628))

Abstract

Extracellular vesicles (EVs) are naturally occurring membranous particles that can be isolated from blood and other biofluids. EVs have drawn considerable attention for their potential as a minimally invasive biomarker source for a range of conditions, based on tissue-specific expression of proteins and other molecular information. To promote robust characterization of EV isolates, the International Society for Extracellular Vesicles (ISEV) has established consensus minimal requirements for the study of extracellular vesicles (MISEV) reporting guidelines. A core element of MISEV guidance is the recommendation for the analysis of protein markers in samples, including positive EV-associated markers and negative contaminant markers based on commonly co-isolated components of the sample matrix. Furthermore, there is growing interest in circulating EVs enriched for tissue-specific origin, and in this context, the degree of nontarget EV “contamination” (e.g., EVs derived from blood cells) may inform assessment of sample purity. The increasing application of EVs as a liquid biopsy for clinical applications requires a high-throughput multiplexed approach that enables analysis of protein markers from small volumes of starting material, ideally utilizing the same platform for measuring biomarkers of interest. To this end, targeted liquid chromatography mass spectrometry using multiple reaction monitoring (LC-MRM-MS) is a key platform for the quantitative assessment of target proteins within EV samples. Here we describe a protocol for the isolation of EVs from blood and parallel analytical methods targeting general EV markers and blood cell-derived EV markers, along with guidance of best practice for sample collection and processing.

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Authors and Affiliations

  1. College of Medicine and Public Health, Flinders University, Adelaide, SA, Australia

    Lauren A. Newman, Zivile Useckaite, Ting Wu, Michael J. Sorich & Andrew Rowland

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  1. Lauren A. Newman
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  2. Zivile Useckaite
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  3. Ting Wu
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  4. Michael J. Sorich
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  5. Andrew Rowland
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Correspondence to Andrew Rowland .

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Editors and Affiliations

  1. Molecular Proteomics, Baker Heart and Diabetes Institute, Melbourne, VIC, Australia

    David W. Greening

  2. Department of Biochemistry and Chemistry, School of Agriculture, Biomedicine and Environment, La Trobe Institute for Molecular Science, Melbourne, VIC, Australia

    Richard J. Simpson

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Newman, L.A., Useckaite, Z., Wu, T., Sorich, M.J., Rowland, A. (2023). Analysis of Extracellular Vesicle and Contaminant Markers in Blood Derivatives Using Multiple Reaction Monitoring. In: Greening, D.W., Simpson, R.J. (eds) Serum/Plasma Proteomics. Methods in Molecular Biology, vol 2628. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2978-9_20

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  • DOI: https://doi.org/10.1007/978-1-0716-2978-9_20

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2977-2

  • Online ISBN: 978-1-0716-2978-9

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