Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins
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Curated by eLife
eLife assessment
This study will be of interest and valuable to scientists of many different subareas in the study of eukaryotic extracellular vesicles. The authors' efforts to improve the way we analyze EVs are highly appreciated and their results are convincing: they not only used appropriate and validated methodologies in line with current state-of-the-art but also presented alternatives to improve these approaches.
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Abstract
Extracellular vesicles (EVs) are released by all cells into biofluids such as plasma. The separation of EVs from highly abundant free proteins and similarly sized lipoproteins remains technically challenging. We developed a digital ELISA assay based on Single Molecule Array (Simoa) technology for ApoB-100, the protein component of several lipoproteins. Combining this ApoB-100 assay with previously developed Simoa assays for albumin and three tetraspanin proteins found on EVs (Ter-Ovanesyan, Norman et al., 2021), we were able to measure the separation of EVs from both lipoproteins and free proteins. We used these five assays to compare EV separation from lipoproteins using size exclusion chromatography with resins containing different pore sizes. We also developed improved methods for EV isolation based on combining several types of chromatography resins in the same column. We present a simple approach to quantitatively measure the main impurities of EV isolation in plasma and apply this approach to develop novel methods for enriching EVs from human plasma. These methods will enable applications where high-purity EVs are required to both understand EV biology and profile EVs for biomarker discovery.
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eLife assessment
This study will be of interest and valuable to scientists of many different subareas in the study of eukaryotic extracellular vesicles. The authors' efforts to improve the way we analyze EVs are highly appreciated and their results are convincing: they not only used appropriate and validated methodologies in line with current state-of-the-art but also presented alternatives to improve these approaches.
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Reviewer #1 (Public Review):
The paper by Dr. Ter-Ovanesyan et. all discussing a very important topic in the field of extracellular vesicles: how to enrich EVs compare to more abundant other circulating particles like lipoproteins, especially VLDL and LDL, which overlap in size and density with EVs and make the purification process challenging. The authors discussed several approaches, including size exclusion chromatography, density-gradient centrifugation, and methods combining charge and size separation. They also proposed the Tri-Mode Chromatography (TMC) method as a good alternative to conventional SEC separation. However, the results provided for the TMC method do not fully support the claim. TEM images provided show the presence of lipoprotein particles at a higher rate than EVs. In addition, proteomics data suggest that …
Reviewer #1 (Public Review):
The paper by Dr. Ter-Ovanesyan et. all discussing a very important topic in the field of extracellular vesicles: how to enrich EVs compare to more abundant other circulating particles like lipoproteins, especially VLDL and LDL, which overlap in size and density with EVs and make the purification process challenging. The authors discussed several approaches, including size exclusion chromatography, density-gradient centrifugation, and methods combining charge and size separation. They also proposed the Tri-Mode Chromatography (TMC) method as a good alternative to conventional SEC separation. However, the results provided for the TMC method do not fully support the claim. TEM images provided show the presence of lipoprotein particles at a higher rate than EVs. In addition, proteomics data suggest that lipoproteins and free proteins are still overrating ones associated with EVs.
The importance of this paper is the code available for an automated device for simultaneous fraction collection, which can be very useful for researchers with limited resources since commercial devices are quite expensive.
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Reviewer #2 (Public Review):
The authors of the current study set out to improve the purity of extracellular vesicles obtained from plasma. A well-described problem is that various means of separating extracellular vesicles from other plasma constituents tend to leave residual impurities such as lipoproteins and free proteins in the final extracellular vesicles preparation. Van Deun and colleagues had previously improved on the size exclusion chromatography approach by adding a second form of chromatography using separation based on charge. The current authors have evaluated that method and another gold standard approach, iodixanol gradient ultracentrifugation, and they have extended the work with the addition of a third form of chromatography. They are building on their prior work on separating albumin from plasma extracellular vesicles.
Reviewer #2 (Public Review):
The authors of the current study set out to improve the purity of extracellular vesicles obtained from plasma. A well-described problem is that various means of separating extracellular vesicles from other plasma constituents tend to leave residual impurities such as lipoproteins and free proteins in the final extracellular vesicles preparation. Van Deun and colleagues had previously improved on the size exclusion chromatography approach by adding a second form of chromatography using separation based on charge. The current authors have evaluated that method and another gold standard approach, iodixanol gradient ultracentrifugation, and they have extended the work with the addition of a third form of chromatography. They are building on their prior work on separating albumin from plasma extracellular vesicles.
A major strength of the paper is that the authors have used complementary methods including a digital immunoassay method and transmission electron microscopy to demonstrate the purity of their sample preparation method. In addition, they have used mass spectrometry to show that they are able to profile hundreds of proteins in their plasma extracellular vesicle sample preparations.
Another major strength of the work is that the authors have taken pains to aid others in reproducing and extending the work. The authors used commercially available human pooled plasma, which is a good decision in terms of reproducibility, compared with a single person's plasma. The authors have explained exactly how to make their new chromatography columns, and they've also explained how to make a manual or an automated apparatus to improve the parallel processing of samples. They explained exactly how to fabricate each apparatus, with computer-aided design files and Raspberry Pi software. I anticipate many others will be able to implement what the authors have done because they shared these resources.
Moreover, the authors have shared the essential data needed to understand and vet their work.
Meanwhile, they shared simple and practical information about the preparation of Sepharose columns to improve the yield of chromatography. They showed that in-column washing with PBS yielded more extracellular vesicles compared with washing Sepharose prior to making the column. This finding should help anyone using size-exclusion chromatography or the more sophisticated combinations of chromatography studied herein.
The major weakness of the method is that it remains unclear to what extent the results of proteomic profiling of these purer plasma extracellular vesicles continue to be confounded by free proteins. This is a problem that will take sustained efforts to resolve, but the authors have built the next piece of the road heading in that direction.
The authors have succeeded in their main aims, albeit without being able to completely rid the sample preparations of lipoproteins, which may or may not be possible.
The results support the authors' conclusions.
This work is going to be useful to the increasing number of researchers who find that circulating extracellular vesicles hold promise for the diagnosis of diseases. In order to find the "signal" within the noise of the complex admixture constituting human plasma, a suitable process for separating vesicles from what I would call impurities is essential. The ability to automate that process while also scaling it up are additional essential components for the extracellular vesicle biomarker field to develop into a clinically useful source of biomarkers. The authors have made progress in each of these areas.
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Reviewer #3 (Public Review):
There is a lack of consensus about the best way to isolate EVs from biofluids, mainly due to EVs being present at low levels in clinically relevant samples and difficult to quantify. As a following study of one previous eLife paper (https://elifesciences.org/articles/70725) from the same group, the authors have extended their Simoa assay to ApoB-100, the major protein component of several lipoproteins. Combining with previously developed Simoa assays, the authors developed a quick framework to quantify EVs, albumin, and lipoproteins on the same platform. Additionally, the authors developed a new EV isolation method that combines two additional resins (i.e., cation-exchange resin and Capto Core 700) as a bottom layer below the SEC layer. Although not greater than the density gradient centrifugation, EVs …
Reviewer #3 (Public Review):
There is a lack of consensus about the best way to isolate EVs from biofluids, mainly due to EVs being present at low levels in clinically relevant samples and difficult to quantify. As a following study of one previous eLife paper (https://elifesciences.org/articles/70725) from the same group, the authors have extended their Simoa assay to ApoB-100, the major protein component of several lipoproteins. Combining with previously developed Simoa assays, the authors developed a quick framework to quantify EVs, albumin, and lipoproteins on the same platform. Additionally, the authors developed a new EV isolation method that combines two additional resins (i.e., cation-exchange resin and Capto Core 700) as a bottom layer below the SEC layer. Although not greater than the density gradient centrifugation, EVs isolated using the newly developed method showed better purity than with SEC alone or dual-mode chromatography. A device automatically running columns in parallel for EV isolation was further developed to increase the throughput and reproducibility of column-based EV isolation. The development of Simoa assay to ApoB-100 and the Tri-Mode Chromatography would be of great relevance to EV studies.
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