Rapid Isolation of Extracellular Vesicles from Blood Plasma with Size-Exclusion Chromatography Followed by Mass Spectrometry-Based Proteomic Profiling
The presented procedure allows rapid isolation of extracellular vesicles (EVs) from plasma using size-exclusion chromatography (SEC). Additionally, an approach for reducing the lipid and salt content of the EV isolate in preparation for mass spectrometry (MS)-based proteomic analysis is presented. An example setup for proteomic profiling of the processed samples by nanoflow liquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS) is also presented. Approximately 1000 protein groups in blood plasma-derived EVs can be identified and quantitated following this procedure and using the described instrumentation.
Key wordsExtracellular vesicles EVs Microparticles Exosomes Proteomics Size-exclusion chromatography Mass spectrometry Nano-LC-MS Sample preparation Proteomic profiling
This work was supported by the ASMS 2015 Research Award (ARI), NIH 1R01GM120272-01 (ARI), and the Dana-Farber Cancer Institute/Northeastern University Joint Seed Funding Program in Cancer Drug Development (ARI). The authors thank Dr. Ionita Ghiran (Beth Israel Deaconess Medical Center) for fruitful discussions and his assistance with blood collection.
- 1.Lotvall J, Hill AF, Hochberg F, Buzas EI, Di Vizio D, Gardiner C, Gho YS, Kurochkin IV, Mathivanan S, Quesenberry P, Sahoo S, Tahara H, Wauben MH, Witwer KW, Thery C (2014) Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles 3:26913. doi: 10.3402/jev.v3.26913 CrossRefPubMedGoogle Scholar
- 2.Gould SJ, Raposo G (2013) As we wait: coping with an imperfect nomenclature for extracellular vesicles. J Extracell Vesicles 2. doi: 10.3402/jev.v2i0.20389
- 6.Tauro BJ, Greening DW, Mathias RA, Ji H, Mathivanan S, Scott AM, Simpson RJ (2012) Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes. Methods 56(2):293–304. doi: 10.1016/j.ymeth.2012.01.002 CrossRefPubMedGoogle Scholar
- 7.Boing AN, van der Pol E, Grootemaat AE, Coumans FA, Sturk A, Nieuwland R (2014) Single-step isolation of extracellular vesicles by size-exclusion chromatography. J Extracell Vesicles 3. doi: 10.3402/jev.v3.23430