Dataset Information

Purification of human plasma exosomes for proteomics: optimization and application to detect changes in response to exercise

ABSTRACT: Extracellular vesicles (EVs) are released into blood from multiple organs and carry molecular cargo that facilitates inter-organ communication and an integrated response to physiological and pathological stimuli. Interrogation of the protein cargo of EVs is currently limited by the absence of optimal and reproducible approaches for purifying plasma EVs that are suitable for downstream proteomic analyses. We describe a size exclusion chromatography (SEC)-based method to purify EVs from platelet poor plasma (PPP) for proteomics profiling via high-resolution mass spectrometry (SEC-MS). The SEC-MS method identified more proteins with higher precision compared to several conventional EV isolation approaches. We applied the SEC-MS method to identify the unique proteomic signatures of EVs released from platelets, adipocytes, muscle cells, and hepatocytes, with the goal of identifying tissue-specific EV markers. Further, we applied the SEC-MS approach to evaluate the effects of a single bout of exercise on EV proteomic cargo in human plasma.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Patrick Vanderboom

LAB HEAD: K Sreekumaran Nair

PROVIDER: PXD026483 | Pride | 2021-08-09

REPOSITORIES: Pride

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A size-exclusion-based approach for purifying extracellular vesicles from human plasma.

Vanderboom Patrick M PM Dasari Surendra S Ruegsegger Gregory N GN Pataky Mark W MW Lucien Fabrice F Heppelmann Carrie Jo CJ Lanza Ian R IR Nair K Sreekumaran KS

Cell reports methods 20210701 3

Extracellular vesicles (EVs) are released into blood from multiple organs and carry molecular cargo that facilitates inter-organ communication and an integrated response to physiological and pathological stimuli. Interrogation of the protein cargo of EVs is currently limited by the absence of optimal and reproducible approaches for purifying plasma EVs that are suitable for downstream proteomic analyses. We describe a size-exclusion chromatography (SEC)-based method to purify EVs from platelet-p ...[more]

PMID: 34355211

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Project description:Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids. Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes. To this end, size exclusion chromatography (SEC) and sucrose density gradient ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque. Effects of post-mortem interval (PMI) before final bdEV separation were probed. MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with macaque, suggesting opportunities for optimization. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Different populations of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease.

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