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

  • Ankit Sinha
  • Simona Principe
  • Javier Alfaro
  • Alex Ignatchenko
  • Vladimir Ignatchenko
  • Thomas KislingerEmail author
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Extracellular vesicles Exosomes Microvesicles Secreted proteins Secretome Proteomics Mass spectrometry 

Notes

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.

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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Ankit Sinha
    • 1
    • 2
  • Simona Principe
    • 2
  • Javier Alfaro
    • 1
    • 2
  • Alex Ignatchenko
    • 2
  • Vladimir Ignatchenko
    • 2
  • Thomas Kislinger
    • 1
    Email author
  1. 1.Department of Medical BiophysicsUniversity of TorontoTorontoCanada
  2. 2.Princess Margaret Cancer CentreTorontoCanada

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