Abstract
Extracellular vesicles (EVs) are nano-sized lipid bilayer surrounded by structures released from most cells, including archaea, bacteria, and eukaryotic cells. EVs play multiple roles in cell-to-cell communication, including immune modulation, angiogenesis, and phenotypic transformation of cells by transferring genetic material and functional proteins. They contain specific subsets of proteins, DNA, RNA, and lipids that represent their cellular origin. Furthermore, EVs are enriched in cell type- or disease-specific proteins, especially plasma membrane proteins, which have pathophysiological functions; many of these vesicular proteins are investigated as novel diagnostic biomarkers, as well as therapeutic targets. To profile the global EV proteome, their various purification methods have been developed, of which density gradient ultracentrifugation is considered especially promising. In this chapter, we describe the isolation of EVs derived from SW480 cells with serum-free media and from U373 cells with EV-depleted serum-containing media, and the preparation of tryptic peptides for mass-spectrometry-based proteomic analysis.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Choi DS, Kim DK, Kim YK, Gho YS (2013) Proteomics, transcriptomics and lipidomics of exosomes and ectosomes. Proteomics 13:1554–1571
Choi DS, Kim DK, Kim YK, Gho YS (2015) Proteomics of extracellular vesicles: exosomes and ectosomes. Mass Spectrom Rev 34:474–490
Kim DK, Kang B, Kim OY, Choi DS, Lee J, Kim SR, Go G, Yoon YJ, Kim JH, Jang SC, Park KS, Choi EJ, Kim KP, Desiderio DM, Kim YK, Lotvall JO, Hwang D, Gho YS (2013) EVpedia: an integrated database of high-throughput data for systemic analyses of extracellular vesicles. J Extracell Vesicles 2:20384
Choi D, Spinelli C, Montermini L, Rak J (2019) Oncogenic regulation of extracellular vesicle proteome and heterogeneity. Proteomics 19:e1800169
Choi D, Montermini L, Kim DK, Meehan B, Roth FP, Rak J (2018) The impact of oncogenic EGFRvIII on the proteome of extracellular vesicles released from glioblastoma cells. Mol Cell Proteomics 17:1948–1964
Thery C, Amigorena S, Raposo G, Clayton A (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol Chapter 3:Unit 3.22
Muralidharan-Chari V, Clancy J, Plou C, Romao M, Chavrier P, Raposo G, D'Souza-Schorey C (2009) ARF6-regulated shedding of tumor cell-derived plasma membrane microvesicles. Curr Biol 19:1875–1885
Kato K, Kobayashi M, Hanamura N, Akagi T, Kosaka N, Ochiya T, Ichik i T (2013) Electrokinetic evaluation of individual exosomes by on-chip microcapillary electrophoresis with laser dark-field microscopy. Jpn J Appl Phys 52:06GK10
Choi DS, Park JO, Jang SC, Yoon YJ, Jung JW, Choi DY, Kim JW, Kang JS, Park J, Hwang D, Lee KH, Park SH, Kim YK, Desiderio DM, Kim KP, Gho YS (2011) Proteomic analysis of microvesicles derived from human colorectal cancer ascites. Proteomics 11:2745–2751
Mathivanan S, Lim JW, Tauro BJ, Ji H, Moritz RL, Simpson RJ (2010) Proteomics analysis of A33 immunoaffinity-purified exosomes released from the human colon tumor cell line LIM1215 reveals a tissue-specific protein signature. Mol Cell Proteomics 9:197–208
Kang D, Oh S, Ahn SM, Lee BH, Moon MH (2008) Proteomic analysis of exosomes from human neural stem cells by flow field-flow fractionation and nanoflow liquid chromatography-tandem mass spectrometry. J Proteome Res 7:3475–3480
Dean WL, Lee MJ, Cummins TD, Schultz DJ, Powell DW (2009) Proteomic and functional characterisation of platelet microparticle size classes. Thromb Haemost 102:711–718
Looze C, Yui D, Leung L, Ingham M, Kaler M, Yao X, Wu WW, Shen RF, Daniels MP, Levine SJ (2009) Proteomic profiling of human plasma exosomes identifies PPARgamma as an exosome-associated protein. Biochem Biophys Res Commun 378:433–438
Hong BS, Cho JH, Kim H, Choi EJ, Rho S, Kim J, Kim JH, Choi DS, Kim YK, Hwang D, Gho YS (2009) Colorectal cancer cell-derived microvesicles are enriched in cell cycle-related mRNAs that promote proliferation of endothelial cells. BMC Genomics 10:556
Wessel D, Flugge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138:141–143
Acknowledgments
We thank to Gyeongyun Go for helping in isolation and characterization of EVs and Jaewook Lee for analysis of the EVs in transmission electron microscope. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2014023004, 2018R1A2A1A05079510, and 2012R1A1A2042534) and by the Foundation Grant (FDN 143322) from Canadian Institutes for Health Research to J.R.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Choi, D., Rak, J., Gho, Y.S. (2021). Isolation of Extracellular Vesicles for Proteomic Profiling. In: Posch, A. (eds) Proteomic Profiling. Methods in Molecular Biology, vol 2261. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1186-9_11
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1186-9_11
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1185-2
Online ISBN: 978-1-0716-1186-9
eBook Packages: Springer Protocols