Assessment of Extracellular Vesicles Purity Using Proteomic Standards

Wang, Tingting; Anderson, Kyle W.; Turko, Illarion V.

doi:10.1021/acs.analchem.7b03119

Cited by 23 publications

(29 citation statements)

References 28 publications

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“…1A and includes two important steps: filtration and SEC. SEC is considered to be a better method for diagnostic assays compared with standard ultracentrifugation as it retains integrity of sEVs ( 13 , 14 ) and concomitantly decreases plasma protein contaminants ( 15 ). Following protocol calibration, the SEC eluent was fractionated into four fractions of 1.5 ml each, and particle numbers were measured by light scattering (NanoSight).…”

Section: Resultsmentioning

confidence: 99%

Proteomic analysis of circulating extracellular vesicles identifies potential markers of breast cancer progression, recurrence, and response

Vinik

Ortega

Mills³

et al. 2020

Sci. Adv.

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Proteomic profiling of circulating small extracellular vesicles (sEVs) represents a promising, noninvasive approach for early detection and therapeutic monitoring of breast cancer (BC). We describe a relatively low-cost, fast, and reliable method to isolate sEVs from plasma of BC patients and analyze their protein content by semiquantitative proteomics. sEV-enriched fractions were isolated from plasma of healthy controls and BC patients at different disease stages before and after surgery. Proteomic analysis of sEV-enriched fractions using reverse phase protein array revealed a signature of seven proteins that differentiated BC patients from healthy individuals, of which FAK and fibronectin displayed high diagnostic accuracy. The size of sEVs was significantly reduced in advanced disease stage, concomitant with a stage-specific protein signature. Furthermore, we observed protein-based distinct clusters of healthy controls, chemotherapy-treated and untreated postsurgery samples, as well as a predictor of high risk of cancer relapse, suggesting that the applied methods warrant development for advanced diagnostics.

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Section: Resultsmentioning

confidence: 99%

Proteomic analysis of circulating extracellular vesicles identifies potential markers of breast cancer progression, recurrence, and response

Vinik

Ortega

Mills³

et al. 2020

Sci. Adv.

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“…Targeted quantitative MS was also used to evaluate the purity of EV preparations. For example, MRM was applied using QconCATs as internal standards for the quantification of EV and non-EV proteins, thus comparing the purity of EVs preparations obtained from human serum by two different isolation protocols [ 141 ].…”

Section: Proteomic Methodsmentioning

confidence: 99%

Proteomics of Extracellular Vesicles: Update on Their Composition, Biological Roles and Potential Use as Diagnostic Tools in Atherosclerotic Cardiovascular Diseases

et al. 2020

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Extracellular vesicles (EVs) are lipid-bound vesicles released from cells under physiological and pathological conditions. Basing on biogenesis, dimension, content and route of secretion, they can be classified into exosomes, microvesicles (MVs) and apoptotic bodies. EVs have a key role as bioactive mediators in intercellular communication, but they are also involved in other physiological processes like immune response, blood coagulation, and tissue repair. The interest in studying EVs has increased over the years due to their involvement in several diseases, such as cardiovascular diseases (CVDs), and their potential role as biomarkers in diagnosis, therapy, and in drug delivery system development. Nowadays, the improvement of mass spectrometry (MS)-based techniques allows the characterization of the EV protein composition to deeply understand their role in several diseases. In this review, a critical overview is provided on the EV’s origin and physical properties, as well as their emerging functional role in both physiological and disease conditions, focusing attention on the role of exosomes in CVDs. The most important cardiac exosome proteomic studies will be discussed giving a qualitative and quantitative characterization of the exosomal proteins that could be used in future as new potential diagnostic markers or targets for specific therapies.

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“…Differential ultracentrifugation (UC) has been a classical method for EV separation, at least until recently [15,16], yet it is time-consuming, labourintensive, and of limited accessibility. To address the obstacles in routine EV extraction, numerous separation techniques based on different principles have been applied for the purification of EVs from biological fluids, such as polymer-based precipitation [17,18], size exclusion chromatography (SEC) [19,20], ultrafiltration (UF) [21], flow field-flow fractionation [22], immunoaffinity capture [23,24], microchip-based techniques [25][26][27] and combinations of these techniques [28,29]. Recently, a number of commercial kits are made available and have been widely reported in the literature for EV isolation.…”

Section: Introductionmentioning

confidence: 99%

Quality and efficiency assessment of six extracellular vesicle isolation methods by nano‐flow cytometry

Tian

Gong

et al. 2019

J of Extracellular Vesicle

428

381

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Extracellular vesicles (EVs) have sparked tremendous interest owing to their prominent potential in diagnostics and therapeutics. Isolation of EVs from complex biological fluids with high purity is essential to the accurate analysis of EV cargo. Unfortunately, generally used isolation techniques do not offer good separation of EVs from non-EV contaminants. Hence, it is important to have a standardized method to characterise the properties of EV preparations, including size distribution, particle concentration, purity and phenotype. Employing a laboratory-built nano-flow cytometer (nFCM) that enables multiparameter analysis of single EVs as small as 40 nm, here we report a new benchmark to the quality and efficiency assessment of EVs isolated from plasma, one of the most difficult body fluids to work with. The performance of five widely used commercial isolation kits was examined and compared with the traditional differential ultracentrifugation (UC). Two to four orders of magnitude higher particle concentrations were observed for EV preparations from platelet-free plasma (PFP) by kits when compared with the EV preparation by UC, yet the purity was much lower. Meanwhile, the particle size distribution profiles of EV preparations by kits closely resembled those of PFP whereas the EV preparation by UC showed a broader size distribution at relatively large particle size. When these kits were used to isolate EVs from vesicle-depleted PFP (VD-PFP), comparable particle counts were obtained with their corresponding EV preparations from PFP, which confirmed again the isolation of a large quantity of non-vesicular contaminants. As CD9, CD63 and CD81 also exist in the plasma matrix, singleparticle phenotyping of EVs offers distinct advantage in the validation of EVs compared with ensemble-averaged approaches, such as Western blot analysis. nFCM allows us to compare different isolation techniques without prejudice.

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Assessment of Extracellular Vesicles Purity Using Proteomic Standards

Cited by 23 publications

References 28 publications

Proteomic analysis of circulating extracellular vesicles identifies potential markers of breast cancer progression, recurrence, and response

Proteomic analysis of circulating extracellular vesicles identifies potential markers of breast cancer progression, recurrence, and response

Proteomics of Extracellular Vesicles: Update on Their Composition, Biological Roles and Potential Use as Diagnostic Tools in Atherosclerotic Cardiovascular Diseases

Quality and efficiency assessment of six extracellular vesicle isolation methods by nano‐flow cytometry

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