Abstract
Exosomes are intercellular messengers with a high potential for diagnostic and therapeutic utility. It is believed that exosomes present in body fluids are responsible for providing signals which inhibit immune cells, interfere with antitumor immunity, and thus influence the response to treatment and its effect. One of the most interesting issues in exosome studies is proper addressing of their cargo composed of nucleic acids and proteins. Effective and selective isolation of extracellular vesicles and identification of proteins present in exosomes has turned out to be a challenging aspect of their exploration. Here we propose a novel approach that is based on isolation of exosomes by mini-size-exclusion chromatography which allows efficient, rapid, and reliable isolation of morphologically intact and functionally active exosomes without the need of ultracentrifugation. The purpose of this chapter is to describe a simple and high-throughput method to isolate, purify, and identify exosomal proteins using a mass spectrometry approach. The proposed protocol compiles the expertise of two research groups specialized in exosome research and in mass spectrometry-based proteomics. The protocol combines differential centrifugation followed by ultrafiltration, centrifugation-based filtration, and gel filtration on Sepharose 2B in order to obtain exosomal fractions characterized by only low contamination with albumin.
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References
Théry C, Clayton A, Amigorena S et al (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol 30:3.22.1–3.22.29
Tickner JA, Urquhart AJ, Stephenson SA et al (2014) Functions and therapeutic roles of exosomes in cancer. Front Oncol 4:127
Zitvogel L, Regnault A, Lozier A et al (1998) Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes. Nat Med 4:594–600
Andre F, Schartz NE, Movassagh M et al (2002) Malignant effusions and immunogenic tumour-derived exosomes. Lancet 360:295–305
Ginestra A, Miceli DL, Dolo V et al (1999) Membrane vesicles in ovarian cancer fluids: a new potential marker. Anticancer Res 19:3439–3445
Adams M, Navabi H, Croston D et al (2005) The rationale for combined chemo/immunotherapy using a Toll-like receptor 3 (TLR3) agonist and tumour-derived exosomes in advanced ovarian cancer. Vaccine 23:2374–2378
Nilsson J, Skog J, Nordstrand A et al (2009) Prostate cancer-derived urine exosomes: a novel approach to biomarkers for prostate cancer. Br J Cancer 100:1603–1607
Abramowicz A, Widlak P, Pietrowska M (2016) Proteomic analysis of exosomal cargo: the challenge of high purity vesicle isolation. Mol Biosyst 12:1407–1419
Wiśniewski JR, Zougman A, Nagaraj N et al (2009) Universal sample preparation method for proteome analysis. Nat Methods 6:359–363
Wiśniewski JR, Zougman A, Mann M (2009) Combination of FASP and StageTip-based fractionation allows in-depth analysis of the hippocampal membrane proteome. J Proteome Res 8:5674–5678
Filter Aided Sample preparation (FASP) Method. http://www.biochem.mpg.de/226356/FASP. Accessed 7 Jun 2016
Tutorials. http://www.biochem.mpg.de/226455/Tutorials. Accessed 7 Jun 2016
Wiśniewski JR, Gaugaz FZ (2015) Fast and sensitive total protein and peptide assays for proteomic analysis. Anal Chem 87:4110–4116
Hong CS, Funk S, Muller L et al (2016) Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer. J Extracell Vesicles 5:29289
Acknowledgements
This work was supported by the National Science Centre, Poland, Grant 2013/11/B/NZ7/01512.
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Pietrowska, M. et al. (2017). Isolation of Exosomes for the Purpose of Protein Cargo Analysis with the Use of Mass Spectrometry. In: Kaufmann, M., Klinger, C., Savelsbergh, A. (eds) Functional Genomics. Methods in Molecular Biology, vol 1654. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7231-9_22
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DOI: https://doi.org/10.1007/978-1-4939-7231-9_22
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Online ISBN: 978-1-4939-7231-9
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