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Results list Most used separation protocols Top EV-enriched proteins

Details	EV-TRACK ID	Experiment nr.	Species	Sample type	separation protocol	First author	Year	EV-METRIC
	EV170028	1/1	Homo sapiens	Synovial fluid	DG dUC SEC	Foers AD	2017	100%
Study summary Full title Enrichment of extracellular vesicles from human synovial fluid using size exclusion chromatography. All authors Foers AD, Chatfield S, Dagley LF, Scicluna BJ, Webb AI, Cheng L, Hill AF, Wicks IP, Pang KC. Journal J Extracell Vesicles Abstract As a complex biological fluid, human synovial fluid (SF) presents challenges for extracellular vesic (show more...)As a complex biological fluid, human synovial fluid (SF) presents challenges for extracellular vesicle (EV) enrichment using standard methods. In this study of human SF, a size exclusion chromatography (SEC)-based method of EV enrichment is shown to deplete contaminants that remain after standard ultracentrifugation-based enrichment methods. Specifically, considerable levels of serum albumin, the high-density lipoprotein marker, apolipoprotein A-I, fibronectin and other extracellular proteins and debris are present in EVs prepared by differential ultracentrifugation. While the addition of a sucrose density gradient purification step improved purification quality, some contamination remained. In contrast, using a SEC-based approach, SF EVs were efficiently separated from serum albumin, apolipoprotein A-I and additional contaminating proteins that co-purified with high-speed centrifugation. Finally, using high-resolution mass spectrometry analysis, we found that residual contaminants which remain after SEC, such as fibronectin and other extracellular proteins, can be successfully depleted by proteinase K. Taken together, our results highlight the limitations of ultracentrifugation-based methods of EV isolation from complex biological fluids and suggest that SEC can be used to obtain higher purity EV samples. In this way, SEC-based methods are likely to be useful for identifying EV-enriched components and improving understanding of EV function in disease. (hide) EV-METRIC 100% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Synovial fluid Sample origin Arthritis Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + dUC + SEC Adj. k-factor 156.9 (pelleting) / 156.9 (washing) Protein markers EV: TSG101/ Rab27b/ Annexin1/ Syntenin/ HSP70/ Flotillin-1 non-EV: Fibronectin/ Albumin/ ApoA1 Proteomics yes Show all info Study aim New methodological development, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Synovial fluid Sample Condition Arthritis Separation Method Differential ultracentrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting: time(min) 90 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Wash: time (min) 90 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 156.9 Density gradient Density medium Sucrose Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 0.25 Highest density fraction 2 Sample volume (mL) 0.65 Orientation Top-down (sample migrates downwards) Rotor type SW 40 Ti Speed (g) 202000 Duration (min) 1100 Fraction volume (mL) 1.05 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: duration (min) 90 Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 122.2 Size-exclusion chromatography Total column volume (mL) 320 Sample volume/column (mL) 13 Resin type Sephacryl S-500 HR Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin-1, HSP70, TSG101, Syntenin, Rab27b, Annexin1 Not detected contaminants Albumin, ApoA1 Proteomics database Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) x EV concentration Yes Particle yield x EM EM-type Transmission-EM Image type Close-up, Wide-field

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