HIV Protocols pp 311-326 | Cite as

Proteomic Characterization of Exosomes from HIV-1-Infected Cells

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


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.

Key words

HIV-1 Exosome Proteomics Mass spectrometry SILAC 


  1. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Sundquist WI, Krausslich HG (2012) HIV-1 assembly, budding, and maturation. Cold Spring Harb Perspect Med 2:a006924. doi:10.1101/cshperspect.a006924 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 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]CrossRefPubMedGoogle Scholar
  4. 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]CrossRefPubMedPubMedCentralGoogle Scholar
  5. 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]CrossRefPubMedGoogle Scholar
  6. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 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]PubMedGoogle Scholar
  9. 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 1:376–386CrossRefPubMedGoogle Scholar
  10. 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]CrossRefPubMedGoogle Scholar
  11. 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 CrossRefPubMedGoogle Scholar
  12. 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 CrossRefPubMedGoogle Scholar
  13. 13.
    Mann M (2006) Functional and quantitative proteomics using SILAC. Nat Rev Mol Cell Biol 7:952–958. doi:10.1038/nrm2067, nrm2067 [pii]CrossRefPubMedGoogle Scholar
  14. 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. 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
  16. 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 CrossRefPubMedGoogle Scholar
  17. 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]CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Mathivanan S, Simpson RJ (2009) ExoCarta: a compendium of exosomal proteins and RNA. Proteomics 9:4997–5000. doi:10.1002/pmic.200900351 CrossRefPubMedGoogle Scholar
  19. 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]CrossRefPubMedPubMedCentralGoogle Scholar
  20. 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]CrossRefPubMedPubMedCentralGoogle Scholar
  21. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 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]CrossRefGoogle Scholar
  23. 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]CrossRefPubMedPubMedCentralGoogle Scholar
  24. 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]CrossRefPubMedGoogle Scholar
  25. 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:R28CrossRefPubMedPubMedCentralGoogle Scholar
  26. 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]CrossRefPubMedGoogle Scholar
  27. 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]CrossRefGoogle Scholar
  28. 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]CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Laboratory of Retrovirology, Division of Infectious Diseases, Department of MedicineThe Warren Alpert Medical School of Brown UniversityProvidenceUSA
  2. 2.Laboratory of Retrovirology, Division of Infectious Diseases, Department of MedicineThe Warren Alpert Medical School of Brown UniversityProvidenceUSA

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