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A Protocol for Isolation and Proteomic Characterization of Distinct Extracellular Vesicle Subtypes by Sequential Centrifugal Ultrafiltration

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Exosomes and Microvesicles

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1545))

Abstract

Scientific and clinical interest in extracellular vesicles (EVs) has increased rapidly as evidence mounts that they may constitute a new signaling paradigm. Recent studies have highlighted EVs carry preassembled complex biological information that elicit pleiotropic responses in target cells. It is well recognized that cells secrete essentially two EV subtypes that can be partially separated by differential centrifugation (DC): the larger size class (referred to as “microvesicles” or “shed microvesicles,” sMVs) is heterogeneous (100–1500 nm), while the smaller size class (referred to as “exosomes”) is relatively homogeneous in size (50–150 nm). A key issue hindering progress in understanding underlying mechanisms of EV subtype biogenesis and cargo selectivity has been the technical challenge of isolating homogeneous EV subpopulations suitable for molecular analysis. In this protocol we reveal a novel method for the isolation, purification, and characterization of distinct EV subtypes: exosomes and sMVs. This method, based on sequential centrifugal ultrafiltration (SCUF), affords unbiased isolation of EVs from conditioned medium from a human colon cancer cell model. For both EV subtypes, this protocol details extensive purification and characterization based on dynamic light scattering, cryoelectron microscopy, quantitation, immunoblotting, and comparative label-free proteome profiling. This analytical SCUF method developed is potentially scalable using tangential flow filtration and provides a solid foundation for future in-depth functional studies of EV subtypes from diverse cell types.

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Acknowledgments

This work was supported, in part, by the La Trobe University Leadership RFA Grant (D.W.G, R.J.S), La Trobe Institute for Molecular Science Molecular Biology Fellowship (D.W.G), and La Trobe University Start-up Grant (D.W.G). RX is supported by La Trobe University Post Graduate Scholarship. We acknowledge the La Trobe University Comprehensive Proteomics Platform.

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Authors and Affiliations

  1. Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science (LIMS), La Trobe University, Room 422, LIMS1, Bundoora, Melbourne, VIC, 3086, Australia

    Rong Xu

  2. Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science (LIMS), La Trobe University, Room 423, LIMS1, Bundoora, Melbourne, VIC, 3086, Australia

    David W. Greening

  3. Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science (LIMS), La Trobe University, Room 414, LIMS1, Bundoora, Melbourne, VIC, 3086, Australia

    Richard J. Simpson

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  1. Rong Xu
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  2. Richard J. Simpson
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Correspondence to David W. Greening .

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Editors and Affiliations

  1. Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia

    Andrew F Hill

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Xu, R., Simpson, R.J., Greening, D.W. (2017). A Protocol for Isolation and Proteomic Characterization of Distinct Extracellular Vesicle Subtypes by Sequential Centrifugal Ultrafiltration. In: Hill, A. (eds) Exosomes and Microvesicles. Methods in Molecular Biology, vol 1545. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6728-5_7

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  • DOI: https://doi.org/10.1007/978-1-4939-6728-5_7

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  • Print ISBN: 978-1-4939-6726-1

  • Online ISBN: 978-1-4939-6728-5

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