Abstract
Membranous vesicles are constitutively released by a multitude of cell types. Following fusion of multivesicular bodies with the plasma membrane, endocytic vesicles, 30–90 nm in size termed exosomes are released extracellularly. Whilst several groups have reported the presence of exosomes in cell-culture conditioned medium, their biological and physiological functions still remain unclear. In addition, exosomes have been detected in body fluids associated with disease, further demonstrating their potential as diagnostic biomarkers. This protocol employs size filtration followed by ultracentrifugation to isolate and purify exosomes from the colon carcinoma cell line LIM 1215. Morphological visualisation and characterisation is based on electron microscopy and western blotting, whilst protein identification is achieved using a combination of 1D SDS-PAGE and LC-MS/MS.
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References
Pan, B. T., and Johnstone, R. M. (1983) Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor. Cell 33, 967–978.
Harding, C., Heuser, J., and Stahl, P. (1983) Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes. J Cell Biol 97, 329–339.
Fevrier, B., and Raposo, G. (2004) Exosomes: endosomal-derived vesicles shipping extracellular messages. Curr Opin Cell Biol 16, 415–421.
Pan, B. T., Teng, K., Wu, C., Adam, M., and Johnstone, R. M. (1985) Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes. J Cell Biol 101, 942–948.
Johnstone, R. M., Adam, M., Hammond, J. R., Orr, L., and Turbide, C. (1987) Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes). J Biol Chem 262, 9412–9420.
Thery, C., Zitvogel, L., and Amigorena, S. (2002) Exosomes: composition, biogenesis and function. Nat Rev Immunol 2, 569–579.
Raposo, G., Nijman, H. W., Stoorvogel, W., Liejendekker, R., Harding, C. V., Melief, C. J., and Geuze, H. J. (1996) B lymphocytes secrete antigen-presenting vesicles. J Exp Med 183, 1161–1172.
Wubbolts, R., Leckie, R. S., Veenhuizen, P. T., Schwarzmann, G., Mobius, W., Hoernschemeyer, J., Slot, J. W., Geuze, H. J., and Stoorvogel, W. (2003) Proteomic and biochemical analyses of human B cell-derived exosomes. Potential implications for their function and multivesicular body formation. J Biol Chem 278, 10963–10972.
Thery, C., Regnault, A., Garin, J., Wolfers, J., Zitvogel, L., Ricciardi-Castagnoli, P., Raposo, G., and Amigorena, S. (1999) Molecular characterization of dendritic cell-derived exosomes. Selective accumulation of the heat shock protein hsc73. J Cell Biol 147, 599–610.
Thery, C., Boussac, M., Veron, P., Ricciardi-Castagnoli, P., Raposo, G., Garin, J., and Amigorena, S. (2001) Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles. J Immunol 166, 7309– 7318.
van Niel, G., Raposo, G., Candalh, C., Boussac, M., Hershberg, R., Cerf-Bensussan, N., and Heyman, M. (2001) Intestinal epithelial cells secrete exosome-like vesicles. Gastroenterology 121, 337–349.
Van Niel, G., Mallegol, J., Bevilacqua, C., Candalh, C., Brugiere, S., Tomaskovic-Crook, E., Heath, J. K., Cerf-Bensussan, N., and Heyman, M. (2003) Intestinal epithelial exosomes carry MHC class II/peptides able to inform the immune system in mice. Gut 52, 1690–1697.
Bard, M. P., Hegmans, J. P., Hemmes, A., Luider, T. M., Willemsen, R., Severijnen, L. A., van Meerbeeck, J. P., Burgers, S. A., Hoogsteden, H. C., and Lambrecht, B. N. (2004) Proteomic analysis of exosomes isolated from human malignant pleural effusions. Am J Respir Cell Mol Biol 31, 114–121.
Mears, R., Craven, R. A., Hanrahan, S., Totty, N., Upton, C., Young, S. L., Patel, P., Selby, P. J., and Banks, R. E. (2004) Proteomic analysis of melanoma-derived exosomes by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Proteomics 4, 4019-4031.
Hegmans, J. P., Bard, M. P., Hemmes, A., Luider, T. M., Kleijmeer, M. J., Prins, J. B., Zitvogel, L., Burgers, S. A., Hoogsteden, H. C., and Lambrecht, B. N. (2004) Proteomic analysis of exosomes secreted by human mesothelioma cells. Am J Pathol 164, 1807–1815.
Pisitkun, T., Shen, R. F., and Knepper, M. A. (2004) Identification and proteomic profiling of exosomes in human urine. Proc Natl Acad Sci USA 101, 13368–13373.
Segura, E., Amigorena, S., and Thery, C. (2005) Mature dendritic cells secrete exosomes with strong ability to induce antigen-specific effector immune responses. Blood Cells Mol Dis 35, 89–93.
Taylor, D. D., and Black, P. H. (1986) Shedding of plasma membrane fragments. Neoplastic and developmental importance. Dev Biol (N Y 1985) 3, 33–57.
Taylor, D. D., Taylor, C. G., Jiang, C. G., and Black, P. H. (1988) Characterization of plasma membrane shedding from murine melanoma cells. Int J Cancer 41, 629–635.
Dolo, V., Adobati, E., Canevari, S., Picone, M. A., and Vittorelli, M. L. (1995) Membrane vesicles shed into the extracellular medium by human breast carcinoma cells carry tumor-associated surface antigens. Clin Exp Metastasis 13, 277–286.
Dolo, V., Ginestra, A., Cassara, D., Violini, S., Lucania, G., Torrisi, M. R., Nagase, H., Canevari, S., Pavan, A., and Vittorelli, M. L. (1998) Selective localization of matrix metalloproteinase 9, beta1 integrins, and human lymphocyte antigen class I molecules on membrane vesicles shed by 8701-BC breast carcinoma cells. Cancer Res 58, 4468–4474.
Hoorn, E. J., Pisitkun, T., Zietse, R., Gross, P., Frokiaer, J., Wang, N. S., Gonzales, P. A., Star, R. A., and Knepper, M. A. (2005) Prospects for urinary proteomics: exosomes as a source of urinary biomarkers. Nephrology (Carlton) 10, 283–290.
Lamparski, H. G., Metha-Damani, A., Yao, J. Y., Patel, S., Hsu, D. H., Ruegg, C., and Le Pecq, J. B. (2002) Production and characterization of clinical grade exosomes derived from dendritic cells. J Immunol Methods 270, 211–226.
Koga, K., Matsumoto, K., Akiyoshi, T., Kubo, M., Yamanaka, N., Tasaki, A., Nakashima, H., Nakamura, M., Kuroki, S., Tanaka, M., and Katano, M. (2005) Purification, characterization and biological significance of tumor-derived exosomes. Anticancer Res 25, 3703–3707.
Moritz, R. L., Ji, H., Schutz, F., Connolly, L. M., Kapp, E. A., Speed, T. P., and Simpson, R. J. (2004) A proteome strategy for fractionating proteins and peptides using continuous free-flow electrophoresis coupled off-line to reversed-phase high-performance liquid chromatography. Anal Chem 76, 4811–4824.
Simpson, R. J., Connolly, L. M., Eddes, J. S., Pereira, J. J., Moritz, R. L., and Reid, G. E. (2000) Proteomic analysis of the human colon carcinoma cell line (LIM 1215): development of a membrane protein database. Electrophoresis 21, 1707–1732.
Phillips, H. J., and Terryberry, J. E. (1957) Counting actively metabolizing tissue cultured cells. Exp Cell Res 13, 341–347.
Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65, 55–63.
Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150, 76–85.
Wolfers, J., Lozier, A., Raposo, G., Regnault, A., Thery, C., Masurier, C., Flament, C., Pouzieux, S., Faure, F., Tursz, T., Angevin, E., Amigorena, S., and Zitvogel, L. (2001) Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming. Nat Med 7, 297–303.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Mathias, R.A., Lim, J.W., Ji, H., Simpson, R.J. (2009). Isolation of Extracellular Membranous Vesicles for Proteomic Analysis. In: Peirce, M.J., Wait, R. (eds) Membrane Proteomics. Methods in Molecular Biology™, vol 528. Humana Press. https://doi.org/10.1007/978-1-60327-310-7_16
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DOI: https://doi.org/10.1007/978-1-60327-310-7_16
Publisher Name: Humana Press
Print ISBN: 978-1-60327-309-1
Online ISBN: 978-1-60327-310-7
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