Proteomic Profiling of Secreted Proteins, Exosomes, and Microvesicles in Cell Culture Conditioned Media
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 wordsExtracellular vesicles Exosomes Microvesicles Secreted proteins Secretome Proteomics Mass spectrometry
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.
- 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 25:2245–2256CrossRefPubMedPubMedCentralGoogle Scholar