Automated On-Line Isolation and Fractionation Method for Subpopulations of Extracellular Vesicles (original) (raw)
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Analytical chemistry, 2020
An automated on-line isolation and fractionation system including controlling software was developed for selected nanosized biomacromolecules from human plasma by on-line coupled immunoaffinity chromatography-asymmetric flow field-flow fractionation (IAC-AsFlFFF). The on-line system was versatile, only different monoclonal antibodies, anti-apolipoprotein B-100, anti-CD9, or anti-CD61, were immobilized on monolithic disk columns for isolation of lipoproteins and extracellular vesicles (EVs). The platelet-derived CD61-positive EVs and CD9-positive EVs, isolated by IAC, were further fractionated by AsFlFFF to their size-based subpopulations (e.g., exomeres and exosomes) for further analysis. Field-emission scanning electron microscopy elucidated the morphology of the subpopulations, and 20 free amino acids and glucose in EV subpopulations were identified and quantified in the ng/mL range using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). The study revealed that there were significant differences between EV origin and size-based subpopulations. The on-line coupled IAC-AsFlFFF system was successfully programmed for reliable execution of 10 sequential isolation and fractionation cycles (37−80 min per cycle) with minimal operator involvement, minimal sample losses, and contamination. The relative standard deviations (RSD) between the cycles for human plasma samples were 0.84−6.6%. H uman biomacromolecules are complex and structurally diverse. They are excellent biomarkers, needed for the recognition and detection of the early stages of diseases, to understand the pathogenesis of the disease, and to find the treatment solutions. Their fast and reliable isolation and purification are often challenging or even a bottleneck for their diagnostic and therapeutic applications. 1 The challenges are caused by their stability requirements and unique nature, especially when isolated from human biofluids. New systems are needed to eliminate the problems, such as operator dependent errors, 2 aggregation, 3,4 oxidation, shear and mechanical stress, and contamination, 5 present in the current methods and techniques, which are often also tedious and time consuming. The study of subpopulations of lipoproteins and extracellular vesicles (EVs) has proven to be important for detection of different diseases 6,7 (e.g., small dense low-density lipoprotein (LDL) particles are considered unfavorable for health 8,9), or for finding a new extracellular vesicle subpopulation called exomere, 10 whose effects on health are still unclear. EV is a general term that includes both ectosomal and endosomal EVs (exosomes). Their size and other biophysical characteristics are similar, and thus EVs and exosomes cannot be separated based on size or density alone. 11 Therefore specific immunobased separation techniques are needed for future clinical diagnostic and therapeutic applications of exo-somes, 6,11,12 and especially the study of subpopulations of EVs has lately attracted great attention. 6,10,13
Detailed analysis of the plasma extracellular vesicle proteome after separation from lipoproteins
Cellular and Molecular Life Sciences
The isolation of extracellular vesicles (EVs) from blood is of great importance to understand the biological role of circulating EVs and to develop EVs as biomarkers of disease. Due to the concurrent presence of lipoprotein particles, however, blood is one of the most difficult body fluids to isolate EVs from. The aim of this study was to develop a robust method to isolate and characterise EVs from blood with minimal contamination by plasma proteins and lipoprotein particles. Plasma and serum were collected from healthy subjects, and EVs were isolated by size-exclusion chromatography (SEC), with most particles being present in fractions 8-12, while the bulk of the plasma proteins was present in fractions 11-28. Vesicle markers peaked in fractions 7-11; however, the same fractions also contained lipoprotein particles. The purity of EVs was improved by combining a density cushion with SEC to further separate lipoprotein particles from the vesicles, which reduced the contamination of lipoprotein particles by 100-fold. Using this novel isolation procedure, a total of 1187 proteins were identified in plasma EVs by mass spectrometry, of which several proteins are known as EV-associated proteins but have hitherto not been identified in the previous proteomic studies of plasma EVs. This study shows that SEC alone is unable to completely separate plasma EVs from lipoprotein particles. However, combining SEC with a density cushion significantly improved the separation of EVs from lipoproteins and allowed for a detailed analysis of the proteome of plasma EVs, thus making blood a viable source for EV biomarker discovery.
Frontiers in Physiology
Background: Extracellular vesicles (EVs) (isolated from blood plasma) are currently being extensively researched, both as biomarkers and for their therapeutic possibilities. One challenging aspect to this research is the efficient isolation of high-purity EVs from blood plasma in quantities sufficient for in vivo experiments. In accordance with this challenge, the aim of this study was to develop an isolation method in which to separate the majority of EVs from major impurities such as lipoprotein particles and the abundant plasma proteins albumin and fibrinogen. Methods: Samples of rat blood were centrifuged to remove cells, platelets, large EVs and protein aggregates without prior filtration. Density gradient ultracentrifugation was performed by loading plasma sample onto 50, 30, and 10% iodixanol layers and then centrifuged at 120,000 × g for 24 h. Ten fractions (F1-10) were collected from top to bottom. Fractions with the highest EV content were further purified by ultracentrifugation, size exclusion, or bind-elute chromatography. Efficiency and purity were assessed by Western blots. Morphology and size distribution of particles were examined by dynamic light scattering and electron microscopy (EM). Results: The highest band intensities of EV markers Alix, Tsg101 and CD81 were detected by Western blot in F6 of small-scale DGUC (61.5 ± 10.4%; 48.1 ± 5.8%; 41.9 ± 3.8%, respectively) at a density of 1.128-1.174 g/mL, where the presence of vesicles with a mean diameter of 38 ± 2 nm was confirmed by EM and DLS. Only 1.4 ± 0.5% of LDL and chylomicron marker, 3.0 ± 1.3% of HDL marker, and 9.9 ± 0.4% of albumin remained in the EV-rich F6. However, 32.8 ± 1.5% of the total fibrinogen beta was found in this fraction. Second-step purification by UC or SEC did not improve EV separation, while after BEC on HiScreen Capto Core 700 albumin and lipoprotein
Journal of Extracellular Vesicles, 2018
Interest in small extracellular vesicles (sEVs) as functional carriers of proteins and nucleic acids is growing continuously. There are large numbers of sEVs in the blood, but lack of standardised methods for sEV isolation greatly limits our ability to study them. In this report, we use rat plasma to systematically compare two commonly used techniques for isolation of sEVs: ultracentrifugation (UC-sEVs) and size-exclusion chromatography (SEC-sEVs). SEC-sEVs had higher particle number, protein content, particle/protein ratios and sEV marker signal than UC-sEVs. However, SEC-sEVs also contained greater amounts of APOB + lipoproteins and large quantities of non-sEV protein. sEV marker signal correlated very well with both particle number and protein content in UC-sEVs but not in all of the SEC-sEV fractions. Functionally, both UC-sEVs and SEC-sEVs isolates contained a variety of proangiogenic factors (with endothelin-1 being the most abundant) and stimulated migration of endothelial cells. However, there was no evident correlation between the promigratory potential and the quantity of sEVs added, indicating that non-vesicular co-isolates may contribute to the promigratory effects. Overall, our findings suggest that UC provides plasma sEVs of lower yields, but markedly higher purity compared to SEC. Furthermore, we show that the functional activity of sEVs can depend on the isolation method used and does not solely reflect the sEV quantity. These findings are of importance when working with sEVs isolated from plasmaor serum-containing conditioned medium.
Scientific Reports, 2020
Human plasma is a complex fluid, increasingly used for extracellular vesicle (EV) biomarker studies. Our aim was to find a simple EV-enrichment method for reliable quantification of EVs in plasma to be used as biomarker of disease. Plasma of ten healthy subjects was processed using sedimentation rate- (sucrose cushion ultracentrifugation—sUC) and size- (size exclusion chromatography—SEC) based methods. According to nanoparticle tracking analysis (NTA), asymmetrical flow field-flow fractionation coupled to detectors (AF4-UV-MALS), miRNA quantification, transmission electron microscopy and enzyme-linked immunosorbent assay, enrichment of EVs from plasma with sUC method lead to high purity of EVs in the samples. High nanoparticle concentrations after SEC resulted from substantial contamination with lipoproteins and other aggregates of EV-like sizes that importantly affect downstream EV quantification. Additionally, sUC EV-enrichment method linked to quantification with NTA or AF4-UV-MA...
RNA Biology, 2019
Ultracentrifugation remains the gold standard for isolation of small extracellular vesicles (sEV), particularly for cancer applications. The objective of this study was to determine if a widely used ultracentrifugation protocol for isolation of serum sEV could be modified to reduce the number of ultracentrifugation cycles and increase efficiency, while maintaining equal or better sample purity and yield. Serum was obtained from two healthy subjects. sEVs were isolated from 1 mL aliquots using three different ultracentrifugation protocols. Co-isolation of RNA carrier protein was assessed by performing Western blots for ApoA-I, ApoB, and Ago2. Small RNA-sequencing was performed on the sEV isolates, and differential detection of small ncRNA was compared across isolation protocols. Reduction from three-to two-ultracentrifuge cycles with no sucrose cushion resulted in a much higher sEV yield but also had the highest levels of lipoprotein and Ago2 contamination. However, the two-ultracentrifugation cycle protocol that incorporated a 30% sucrose cushion into the first cycle resulted in slightly higher sEV yields with lower levels of protein contamination compared to the lengthier three-ultracentrifugation cycle approach, therefore presenting a more efficient alternative approach for isolation of serum sEVs. It was also notable that there were some differences in sEV ncRNA cargo according to protocol, although it was less than expected given the differences in co-isolated RNA carrier proteins. Our results suggest that use of the modified serum sEV isolation protocol with two ultracentrifugation cycles and incorporating a 30% sucrose cushion offers a more efficient approach in terms of efficiency and purity.
Lipidomic characterization of extracellular vesicles in human serum
Journal of Circulating Biomarkers
There is a wide variety of extracellular vesicles (EVs) that differ in size and cargo composition. EVs isolated from human plasma or serum carry lipid, protein, and RNA cargo that provides insights to the regulation of normal physiological processes, and to pathological states. Specific populations of EVs have been proposed to contain protein and RNA cargo that are biomarkers for neurologic and systemic diseases. Although there is a considerable amount of evidence that circulating lipids are biomarkers for multiple disease states, it not clear if these lipid biomarkers are enriched in EVs, or if specific populations of EVs are enriched for particular classes of lipid. A highly reproducible workflow for the analysis of lipid content in EVs isolated from human plasma or serum would facilitate this area of research. Here we optimized an MS/MSALL workflow for the untargeted analysis of the lipid content in EVs isolated from human serum. A simple sequential ultracentrifugation protocol i...
Human Serum Extracellular Vesicle Proteomic Profile Depends on the Enrichment Method Employed
International Journal of Molecular Sciences, 2021
The proteomic profiling of serum samples supposes a challenge due to the large abundance of a few blood proteins in comparison with other circulating proteins coming from different tissues and cells. Although the sensitivity of protein detection has increased enormously in the last years, specific strategies are still required to enrich less abundant proteins and get rid of abundant proteins such as albumin, lipoproteins, and immunoglobulins. One of the alternatives that has become more promising is to characterize circulating extracellular vesicles from serum samples that have great interest in biomedicine. In the present work, we enriched the extracellular vesicles fraction from human serum by applying different techniques, including ultracentrifugation, size-exclusion chromatography, and two commercial precipitation methods based on different mechanisms of action. To improve the performance and efficacy of the techniques to promote purity of the preparations, we have employed a s...
International Journal of Molecular Sciences
Extracellular vesicles (EVs) are reminiscent of their cell of origin and thus represent a valuable source of biomarkers. However, for EVs to be used as biomarkers in clinical practice, simple, comparable, and reproducible analytical methods must be applied. Although progress is being made in EV separation methods for human biofluids, the implementation of EV assays for clinical diagnosis and common guidelines are still lacking. We conducted a comprehensive analysis of established EV separation techniques from human serum and plasma, including ultracentrifugation and size exclusion chromatography (SEC), followed by concentration using (a) ultracentrifugation, (b) ultrafiltration, or (c) precipitation, and immunoaffinity isolation. We analyzed the size, number, protein, and miRNA content of the obtained EVs and assessed the functional delivery of EV cargo. Our results demonstrate that all methods led to an adequate yield of small EVs. While no significant difference in miRNA content w...
Annals of Laboratory Medicine, 2020
Methods for reproducibly isolating and enriching small extracellular vesicles (EVs) from blood are essential for clinical utilization of small EVs in cancer patients. We combined ultracentrifugation (UC) with polymer-based precipitation (ExoQuick [EQ] or Total Exosome Isolation [TEI] kit) to isolate small EVs (diameter, 30-150 nm) from the serum of breast cancer patients. We compared the performance of four cycles of UC (UC4x) with that of two cycles of UC followed by enrichment using the EQ (UC2x→EQ) or TEI (UC2x→TEI) kits. The mean concentration of small EVs isolated from 1 mL of serum using UC2x→EQ (139.0 ± 29.1 μg) and UC2x→TEI (140.4 ± 5.0 μg) did not differ from that obtained using UC4x (141.8 ± 26.9 μg). The mean number of EV particles obtained using UC4x was 29.2 ± 9.9 × 10 9 per mL of serum, whereas UC2x→EQ and UC2x→TEI yielded higher numbers of EVs (50.7 ± 17.0 × 10 9 and 59.3 ± 20.6 × 10 9 , respectively). Concentrations of EV micro-RNAs, including miR-21 and miR-155, did not differ between the three methods. In conclusion, performing UC prior to the use of polymer-based precipitation kits could be feasible for isolating small EVs from human serum in large sample-based translational researches.