Harmonization of exosome isolation from culture supernatants for optimized proteomics analysis

Abstract

Exosomes, the smallest subset of extracellular vesicles (EVs), have recently attracted much attention in the scientific community. Their involvement in intercellular communication and molecular reprogramming of different cell types created a demand for a stringent characterization of the proteome which exosomes carry and deliver to recipient cells. Mass spectrometry (MS) has been extensively used for exosome protein profiling. Unfortunately, no standards have been established for exosome isolation and their preparation for MS, leading to accumulation of artefactual data. These include the presence of high-abundance exosome-contaminating serum proteins in culture media which mask low-abundance exosome-specific components, isolation methods that fail to yield “pure” vesicles or variability in protein solubilization protocols. There is an unmet need for the development of standards for exosome generation, harvesting, and isolation from cellular supernatants and for optimization of protein extraction methods before proteomics analysis by MS. In this communication, we illustrate the existing problems in this field and provide a set of recommendations that are expected to harmonize exosome processing for MS and provide the faithful picture of the proteomes carried by exosomes. The recommended workflow for effective and specific identification of proteins in exosomes released by the low number of cells involves culturing cells in medium with a reduced concentration of exosome-depleted serum, purification of exosomes by size-exclusion chromatography, a combination of different protein extraction method and removal of serum-derived proteins from the final dataset using an appropriate sample of cell-unexposed medium as a control. Application of this method allowed detection of >250 vesicle-specific proteins in exosomes from 10 mL of culture medium.

Proteome Profiling of Exosomes Purified from a Small Amount of Human Serum: The Problem of Co-Purified Serum Components

“…We have previously reported a proteomics pipeline optimized for identification of exosome proteins purified by the SEC from the cell culture media [30], which allowed to identify above 1200 proteins in EVs released to media by head and neck cancer cell line, including typical exosome markers (CD9, CD63, CD81, ALIX, TSG101, etc.) and excluding co-purified serum proteins from fetal bovine serum complementing culture media [40].…”

“…However, such combined procedures are more laborious and require a large amount of biomaterial, which could be a disadvantage in routine applications. Here we aimed to characterize the feasibility of shotgun proteomics profiling of exosomes purified from a small amount of serum (<1mL) by the micro-SEC approach successfully implemented in a proteomics study of exosomes released in vitro to cell culture media [30].…”

Low-Vacuum Filtration as an Alternative Extracellular Vesicle Concentration Method: A Comparison with Ultracentrifugation and Differential Centrifugation

“…The interest in extracellular vesicles has grown over the last years [ 31 ], mostly because of their involvement in cell-to-cell communication [ 32 ], cancer progression [ 33 ] and immunosuppression [ 34 ], and due to their great potential as DDS [ 15 , 35 ]. Besides the recent intense development in EV isolation micromethods, there is an unmet need to develop more efficient and repeatable EV isolation and concentration methods for application with high-volume sources, suitable not only for proteomic analysis but also for drug delivery purposes [ 35 , 36 ]. In this study, we have developed and validated a system that is dedicated to EV concentration from high-volume sources of conditioned media: low-vacuum filtration (LVF).…”

Tasmanian devil facial tumor-derived extracellular vesicles reveal mesenchymal transition markers and adhesion molecules related to metastasis

“…EV proteins were extracted according to the method of Abramowicz, et al (26). EV samples were mixed with acetonitrile to a final concentration of 50% (v/v) and evaporated using a centrifugal vacuum concentrator.…”