Skip to main content
SpringerLink
Log in
Book cover

The Surfaceome pp 91–102Cite as

Proteomic Profiling of Secreted Proteins, Exosomes, and Microvesicles in Cell Culture Conditioned Media

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1722))

Abstract

Secreted proteins are of tremendous biological interest since they can act as ligands for receptors to activate downstream signalling cascades or be used as biomarkers if altered abundance is correlated with a specific pathological state. Proteins can be secreted either as soluble molecules or as part of extracellular vesicles (i.e., exosomes or microvesicles). The complete proteomic profiling of secretomes requires analysis of secreted proteins and extracellular vesicles. Hence, the method described here enriches for microvesicles, exosomes, and secreted proteins from conditioned media using differential centrifugation. The three fractions are then analyzed by mass spectrometry-based proteomics for in-depth characterization and comparison of the protein secretome of cell lines.

This is a preview of subscription content, log in via an institution.

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674

    Article  CAS  PubMed  Google Scholar 

  2. Tlsty TD, Coussens LM (2006) Tumor stroma and regulation of cancer development. Annu Rev Pathol 1:119–150

    Article  CAS  PubMed  Google Scholar 

  3. Schiarea S, Solinas G, Allavena P, Scigliuolo GM, Bagnati R, Fanelli R, Chiabrando C (2010) Secretome analysis of multiple pancreatic cancer cell lines reveals perturbations of key functional networks. J Proteome Res 9:4376–4392

    Article  CAS  PubMed  Google Scholar 

  4. Xu BJ, Yan W, Jovanovic B, An AQ, Cheng N, Aakre ME et al (2010) Quantitative analysis of the secretome of TGF-beta signaling-deficient mammary fibroblasts. Proteomics 10:2458–2470

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Mathias RA, Wang B, Ji H, Kapp EA, Moritz RL, Zhu HJ, Simpson RJ (2009) Secretome-based proteomic profiling of Ras-transformed MDCK cells reveals extracellular modulators of epithelial-mesenchymal transition. J Proteome Res 8:2827–2837

    Article  CAS  PubMed  Google Scholar 

  6. Luo X, Liu Y, Wang R, Hu H, Zeng R, Chen H (2011) A high-quality secretome of A549 cells aided the discovery of C4b-binding protein as a novel serum biomarker for non-small cell lung cancer. J Proteome 74:528–538

    Article  CAS  Google Scholar 

  7. Oksvold MP, Neurauter A, Pedersen KW (2015) Magnetic bead-based isolation of exosomes. Methods Mol Biol 1218:465–481

    Article  CAS  PubMed  Google Scholar 

  8. Thery C, Zitvogel L, Amigorena S (2002) Exosomes: composition, biogenesis and function. Nat Rev Immunol 2:569–579

    CAS  PubMed  Google Scholar 

  9. Huang X, Yuan T, Tschannen M, Sun Z, Jacob H, Du M et al (2013) Characterization of human plasma-derived exosomal RNAs by deep sequencing. BMC Genomics 14:319

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Yoon YJ, Kim OY, Gho YS (2014) Extracellular vesicles as emerging intercellular communicasomes. BMB Rep 47:531–539

    Article  PubMed  PubMed Central  Google Scholar 

  11. Beach A, Zhang HG, Ratajczak MZ, Kakar SS (2014) Exosomes: an overview of biogenesis, composition and role in ovarian cancer. J Ovarian Res 7(1):14

    Article  PubMed  PubMed Central  Google Scholar 

  12. Raposo G, Stoorvogel W (2013) Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 200:373–383

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Saleem SN, Abdel-Mageed AB (2014) Tumor-derived exosomes in oncogenic reprogramming and cancer progression. Cell Mol Life Sci 72:1–10

    Article  PubMed  PubMed Central  Google Scholar 

  14. Peinado H, Aleckovic M, Lavotshkin S, Matei I, Costa-Silva B, Moreno-Bueno G et al (2012) Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET. Nat Med 18:883–891

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lu M, Huang B, Hanash SM, Onuchic JN, Ben-Jacob E (2014) Modeling putative therapeutic implications of exosome exchange between tumor and immune cells. Proc Natl Acad Sci U S A 7:E4165–E4174

    Article  Google Scholar 

  16. Lundholm M, Schroder M, Nagaeva O, Baranov V, Widmark A, Mincheva-Nilsson L, Wikstrom P (2014) Prostate tumor-derived exosomes down-regulate NKG2D expression on natural killer cells and CD8+ T cells: mechanism of immune evasion. PLoS One 9:e108925

    Article  PubMed  PubMed Central  Google Scholar 

  17. Luga V, Zhang L, Viloria-Petit AM, Ogunjimi AA, Inanlou MR, Chiu E et al (2012) Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration. Cell 151:1542–1556

    Article  CAS  PubMed  Google Scholar 

  18. Shimoda M, Principe S, Jackson HW, Luga V, Fang H, Molyneux SD et al (2014) Loss of the Timp gene family is sufficient for the acquisition of the CAF-like cell state. Nat Cell Biol 16:889–901

    Article  CAS  PubMed  Google Scholar 

  19. Valenti R, Huber V, Filipazzi P, Pilla L, Sovena G, Villa A et al (2006) Human tumor-released microvesicles promote the differentiation of myeloid cells with transforming growth factor-beta-mediated suppressive activity on T lymphocytes. Cancer Res 66:9290–9298

    Article  CAS  PubMed  Google Scholar 

  20. Aliotta JM, Sanchez-Guijo FM, Dooner GJ, Johnson KW, Dooner MS, Greer KA et al (2007) Alteration of marrow cell gene expression, protein production, and engraftment into lung by lung-derived microvesicles: a novel mechanism for phenotype modulation. Stem Cells 25:2245–2256

    Article  PubMed  PubMed Central  Google Scholar 

  21. Sinha A, Ignatchenko V, Ignatchenko A, Mejia-Guerrero S, Kislinger T (2014) In-depth proteomic analyses of ovarian cancer cell line exosomes reveals differential enrichment of functional categories compared to the NCI 60 proteome. Biochem Biophys Res Commun 445:694–701

    Article  CAS  PubMed  Google Scholar 

  22. Lee TH, D’Asti E, Magnus N, Al-Nedawi K, Meehan B, Rak J (2011) Microvesicles as mediators of intercellular communication in cancer--the emerging science of cellular ‘debris’. Semin Immunopathol 33:455–467

    Article  PubMed  Google Scholar 

  23. Baietti MF, Zhang Z, Mortier E, Melchior A, Degeest G, Geeraerts A et al (2012) Syndecan-syntenin-ALIX regulates the biogenesis of exosomes. Nat Cell Biol 14:677–685

    Article  CAS  PubMed  Google Scholar 

  24. Ebert MP, Korc M, Malfertheiner P, Rocken C (2006) Advances, challenges, and limitations in serum-proteome-based cancer diagnosis. J Proteome Res 5:19–25

    Article  CAS  PubMed  Google Scholar 

  25. Eichelbaum K, Winter M, Berriel Diaz M, Herzig S, Krijgsveld J (2012) Selective enrichment of newly synthesized proteins for quantitative secretome analysis. Nat Biotechnol 30:984–990

    Article  CAS  PubMed  Google Scholar 

  26. 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

    Article  CAS  PubMed  Google Scholar 

  27. Deutsch EW, Mendoza L, Shteynberg D, Farrah T, Lam H, Tasman N et al (2010) A guided tour of the Trans-Proteomic Pipeline. Proteomics 10:1150–1159

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Zhang J, Xin L, Shan B, Chen W, Xie M, Yuen D et al (2012) PEAKS DB: de novo sequencing assisted database search for sensitive and accurate peptide identification. Mol Cell Proteomics 11:M111.010587

    Article  PubMed  Google Scholar 

Download references

Acknowledgment

This study was funded by the Canadian Institutes of Health Research (CIHR) to T.K. (MOP-133615). A.S. was supported through a CIHR Doctoral Award. S.P. was supported by the CIHR Terry Fox Foundation Strategic Training Initiative for Excellence in Radiation Research in the 21st Century. A.I. was supported in parts by the PMH Head and Neck Translational group. T.K. is supported by the Canada Research Chair Program. Support is also provided from the Campbell Family Institute for Cancer Research and the Ministry of Health and Long-term Planning.

Author information

Authors and Affiliations

  1. Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada

    Ankit Sinha, Javier Alfaro & Thomas Kislinger

  2. Princess Margaret Cancer Centre, Toronto, ON, Canada

    Ankit Sinha, Simona Principe, Javier Alfaro, Alex Ignatchenko & Vladimir Ignatchenko

Authors
  1. Ankit Sinha
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Simona Principe
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Javier Alfaro
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alex Ignatchenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Vladimir Ignatchenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Thomas Kislinger
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Thomas Kislinger .

Editor information

Editors and Affiliations

  1. School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, China

    Kenneth R. Boheler

  2. Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin, USA

    Rebekah L. Gundry

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Sinha, A., Principe, S., Alfaro, J., Ignatchenko, A., Ignatchenko, V., Kislinger, T. (2018). Proteomic Profiling of Secreted Proteins, Exosomes, and Microvesicles in Cell Culture Conditioned Media. In: Boheler, K., Gundry, R. (eds) The Surfaceome. Methods in Molecular Biology, vol 1722. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7553-2_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7553-2_6

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7551-8

  • Online ISBN: 978-1-4939-7553-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation