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Proteomic Characterization of Exosomes from HIV-1-Infected Cells

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HIV Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1354))

Abstract

Proteomics has increasingly become an invaluable tool to characterize proteomes from various subcellular compartments. Here, we describe a quantitative proteomics method using the technique of Stable Isotope Labeling by Amino acids in Cell culture (SILAC) to analyze the effects of HIV infection on host exosomal proteomes. The procedure, described below, involves differential isotope labeling of cells, exosome purification, mass spectrometric quantification, and various bioinformatic analyses/verifications. Although this chapter focuses on analyzing the effects of HIV-1 infection on the exosomal proteome, the protocol can easily be adapted to other subcellular compartments under different stress conditions.

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References

  1. Ivanchenko S, Godinez WJ, Lampe M, Krausslich HG, Eils R, Rohr K, Brauchle C, Muller B, Lamb DC (2009) Dynamics of HIV-1 assembly and release. PLoS Pathog 5:e1000652. doi:10.1371/journal.ppat.1000652

    Article  PubMed  PubMed Central  Google Scholar 

  2. Sundquist WI, Krausslich HG (2012) HIV-1 assembly, budding, and maturation. Cold Spring Harb Perspect Med 2:a006924. doi:10.1101/cshperspect.a006924

    Article  PubMed  PubMed Central  Google Scholar 

  3. Keller S, Sanderson MP, Stoeck A, Altevogt P (2006) Exosomes: from biogenesis and secretion to biological function. Immunol Lett 107:102–108. doi:10.1016/j.imlet.2006.09.005, S0165-2478(06)00228-8 [pii]

    Article  CAS  PubMed  Google Scholar 

  4. Schorey JS, Bhatnagar S (2008) Exosome function: from tumor immunology to pathogen biology. Traffic 9:871–881. doi:10.1111/j.1600-0854.2008.00734.x, TRA734 [pii]

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Thery C, Ostrowski M, Segura E (2009) Membrane vesicles as conveyors of immune responses. Nat Rev Immunol 9:581–593. doi:10.1038/nri2567, nri2567 [pii]

    Article  CAS  PubMed  Google Scholar 

  6. Booth AM, Fang Y, Fallon JK, Yang JM, Hildreth JE, Gould SJ (2006) Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane. J Cell Biol 172:923–935. doi:10.1083/jcb.200508014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Van Engelenburg SB, Shtengel G, Sengupta P, Waki K, Jarnik M, Ablan SD, Freed EO, Hess HF, Lippincott-Schwartz J (2014) Distribution of ESCRT machinery at HIV assembly sites reveals virus scaffolding of ESCRT subunits. Science 343:653–656. doi:10.1126/science.1247786

    Article  PubMed  PubMed Central  Google Scholar 

  8. Thery C, Zitvogel L, Amigorena S (2002) Exosomes: composition, biogenesis and function. Nat Rev Immunol 2:569–579. doi:10.1038/nri855, nri855 [pii]

    CAS  PubMed  Google Scholar 

  9. Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1:376–386

    Article  CAS  PubMed  Google Scholar 

  10. Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, Khainovski N, Pillai S, Dey S, Daniels S, Purkayastha S, Juhasz P, Martin S, Bartlet-Jones M, He F, Jacobson A, Pappin DJ (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3:1154–1169. doi:10.1074/mcp.M400129-MCP200, M400129-MCP200 [pii]

    Article  CAS  PubMed  Google Scholar 

  11. Anderson L, Hunter CL (2006) Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins. Mol Cell Proteomics 5:573–588. doi:10.1074/mcp.M500331-MCP200

    Article  CAS  PubMed  Google Scholar 

  12. Nikolov M, Schmidt C, Urlaub H (2012) Quantitative mass spectrometry-based proteomics: an overview. Methods Mol Biol 893:85–100. doi:10.1007/978-1-61779-885-6_7

    Article  CAS  PubMed  Google Scholar 

  13. Mann M (2006) Functional and quantitative proteomics using SILAC. Nat Rev Mol Cell Biol 7:952–958. doi:10.1038/nrm2067, nrm2067 [pii]

    Article  CAS  PubMed  Google Scholar 

  14. Cepko C, Pear W (2001) Retrovirus infection of cells in vitro and in vivo. Curr Protoc Mol Biol Chapter 9: Unit9 14. doi:10.1002/0471142727.mb0914s36

  15. 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. doi: 10.1002/0471143030.cb0322s30

    Google Scholar 

  16. Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26:1367–1372. doi:10.1038/nbt.1511

    Article  CAS  PubMed  Google Scholar 

  17. Li M, Aliotta JM, Asara JM, Wu Q, Dooner MS, Tucker LD, Wells A, Quesenberry PJ, Ramratnam B (2010) Intercellular transfer of proteins as identified by stable isotope labeling of amino acids in cell culture. J Biol Chem 285:6285–6297. doi:10.1074/jbc.M109.057943, M109.057943 [pii]

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Mathivanan S, Simpson RJ (2009) ExoCarta: a compendium of exosomal proteins and RNA. Proteomics 9:4997–5000. doi:10.1002/pmic.200900351

    Article  CAS  PubMed  Google Scholar 

  19. Mathivanan S, Fahner CJ, Reid GE, Simpson RJ (2012) ExoCarta 2012: database of exosomal proteins, RNA and lipids. Nucleic Acids Res 40:D1241–D1244. doi:10.1093/nar/gkr828, gkr828 [pii]

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Fu W, Sanders-Beer BE, Katz KS, Maglott DR, Pruitt KD, Ptak RG (2009) Human immunodeficiency virus type 1, human protein interaction database at NCBI. Nucleic Acids Res 37:D417–D422. doi:10.1093/nar/gkn708, gkn708 [pii]

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Bhatia VN, Perlman DH, Costello CE, McComb ME (2009) Software tool for researching annotations of proteins: open-source protein annotation software with data visualization. Anal Chem 81:9819–9823. doi:10.1021/ac901335x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. da Huang W, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4:44–57. doi:10.1038/nprot.2008.211, nprot.2008.211 [pii]

    Article  CAS  Google Scholar 

  23. Szklarczyk D, Franceschini A, Kuhn M, Simonovic M, Roth A, Minguez P, Doerks T, Stark M, Muller J, Bork P, Jensen LJ, von Mering C (2011) The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Res 39:D561–D568. doi:10.1093/nar/gkq973, gkq973 [pii]

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Cantin R, Diou J, Belanger D, Tremblay AM, Gilbert C (2008) Discrimination between exosomes and HIV-1: purification of both vesicles from cell-free supernatants. J Immunol Methods 338:21–30. doi:10.1016/j.jim.2008.07.007, S0022-1759(08)00216-0 [pii]

    Article  CAS  PubMed  Google Scholar 

  25. Zeeberg BR, Feng W, Wang G, Wang MD, Fojo AT, Sunshine M, Narasimhan S, Kane DW, Reinhold WC, Lababidi S, Bussey KJ, Riss J, Barrett JC, Weinstein JN (2003) GoMiner: a resource for biological interpretation of genomic and proteomic data. Genome Biol 4:R28

    Article  PubMed  PubMed Central  Google Scholar 

  26. Mi H, Muruganujan A, Casagrande JT, Thomas PD (2013) Large-scale gene function analysis with the PANTHER classification system. Nat Protoc 8:1551–1566. doi:10.1038/nprot.2013.092, nprot.2013.092 [pii]

    Article  PubMed  Google Scholar 

  27. Eden E, Navon R, Steinfeld I, Lipson D, Yakhini Z (2009) GOrilla: a tool for discovery and visualization of enriched GO terms in ranked gene lists. BMC Bioinform 10:48. doi:10.1186/1471-2105-10-48, 1471-2105-10-48 [pii]

    Article  Google Scholar 

  28. Kamburov A, Wierling C, Lehrach H, Herwig R (2009) ConsensusPathDB--a database for integrating human functional interaction networks. Nucleic Acids Res 37:D623–D628. doi:10.1093/nar/gkn698, gkn698 [pii]

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by an ARRA supplement to the Lifespan/Tufts/Brown CFAR, P30AI042853-13S1, NIH P20GM103421, P01AA019072, R01HD072693 to BR. This work was also supported by Lifespan Pilot Research Fund (#701-5857), Rhode Island Foundation Medical Research Grant (#20133969), and NIH COBRE URI/RIH Pilot Research Grant (P20GM104317) to ML.

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Correspondence to Ming Li Ph.D. or Bharat Ramratnam M.D. .

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Li, M., Ramratnam, B. (2016). Proteomic Characterization of Exosomes from HIV-1-Infected Cells. In: Prasad, V., Kalpana, G. (eds) HIV Protocols. Methods in Molecular Biology, vol 1354. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3046-3_21

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  • DOI: https://doi.org/10.1007/978-1-4939-3046-3_21

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3045-6

  • Online ISBN: 978-1-4939-3046-3

  • eBook Packages: Springer Protocols

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