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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200081	1/4	Homo sapiens	Blood plasma	DG SEC (non-commercial)	Vergauwen, Glenn	2021	88%
Study summary Full title Robust sequential biophysical fractionation of blood plasma to study variations in the biomolecular landscape of systemically circulating extracellular vesicles across clinical conditions All authors Glenn Vergauwen, Joeri Tulkens, Cláudio Pinheiro, Francisco Avila Cobos, Sándor Dedeyne, Marie-Angélique De Scheerder, Linos Vandekerckhove, Francis Impens, Ilkka Miinalainen, Geert Braems, Kris Gevaert, Pieter Mestdagh, Jo Vandesompele, Hannelore Denys, Olivier De Wever, An Hendrix Journal J Extracell Vesicles Abstract Separating extracellular vesicles (EV) from blood plasma is challenging and complicates their biolog (show more...)Separating extracellular vesicles (EV) from blood plasma is challenging and complicates their biological understanding and biomarker development. In this study, we fractionate blood plasma by combining size-exclusion chromatography (SEC) and OptiPrep density gradient centrifugation to study clinical context-dependent and time-dependent variations in the biomolecular landscape of systemically circulating EV. Using pooled blood plasma samples from breast cancer patients, we first demonstrate the technical repeatability of blood plasma fractionation. Using serial blood plasma samples from HIV and ovarian cancer patients (n = 10) we next show that EV carry a clinical context-dependent and/or time-dependent protein and small RNA composition, including miRNA and tRNA. In addition, differential analysis of blood plasma fractions provides a catalogue of putative proteins not associated with systemically circulating EV. In conclusion, the implementation of blood plasma fractionation allows to advance the biological understanding and biomarker development of systemically circulating EV. (hide) EV-METRIC 88% (99th 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. For the quantitative method, the reporting of measured EV concentration is expected. 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 Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient Size-exclusion chromatography (non-commercial) Protein markers EV: Flotillin1/ CD9 non-EV: APOB/ APOA1 Proteomics no EV density (g/ml) 1.09-1.10 Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1 Not detected contaminants APOA1 ELISA Antibody details provided? No Detected EV-associated proteins CD9 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 50-250

	EV200081	2/4	Homo sapiens	Blood plasma	DG SEC (non-commercial)	Vergauwen, Glenn	2021	50%
Study summary Full title Robust sequential biophysical fractionation of blood plasma to study variations in the biomolecular landscape of systemically circulating extracellular vesicles across clinical conditions All authors Glenn Vergauwen, Joeri Tulkens, Cláudio Pinheiro, Francisco Avila Cobos, Sándor Dedeyne, Marie-Angélique De Scheerder, Linos Vandekerckhove, Francis Impens, Ilkka Miinalainen, Geert Braems, Kris Gevaert, Pieter Mestdagh, Jo Vandesompele, Hannelore Denys, Olivier De Wever, An Hendrix Journal J Extracell Vesicles Abstract Separating extracellular vesicles (EV) from blood plasma is challenging and complicates their biolog (show more...)Separating extracellular vesicles (EV) from blood plasma is challenging and complicates their biological understanding and biomarker development. In this study, we fractionate blood plasma by combining size-exclusion chromatography (SEC) and OptiPrep density gradient centrifugation to study clinical context-dependent and time-dependent variations in the biomolecular landscape of systemically circulating EV. Using pooled blood plasma samples from breast cancer patients, we first demonstrate the technical repeatability of blood plasma fractionation. Using serial blood plasma samples from HIV and ovarian cancer patients (n = 10) we next show that EV carry a clinical context-dependent and/or time-dependent protein and small RNA composition, including miRNA and tRNA. In addition, differential analysis of blood plasma fractions provides a catalogue of putative proteins not associated with systemically circulating EV. In conclusion, the implementation of blood plasma fractionation allows to advance the biological understanding and biomarker development of systemically circulating EV. (hide) EV-METRIC 50% (83rd 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. For the quantitative method, the reporting of measured EV concentration is expected. 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 Blood plasma Sample origin breast cancer Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: None Proteomics yes EV density (g/ml) 1.09-1.10 Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Proteomics database Yes: Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV200081	3/4	Homo sapiens	Blood plasma	DG SEC (non-commercial)	Vergauwen, Glenn	2021	50%
Study summary Full title Robust sequential biophysical fractionation of blood plasma to study variations in the biomolecular landscape of systemically circulating extracellular vesicles across clinical conditions All authors Glenn Vergauwen, Joeri Tulkens, Cláudio Pinheiro, Francisco Avila Cobos, Sándor Dedeyne, Marie-Angélique De Scheerder, Linos Vandekerckhove, Francis Impens, Ilkka Miinalainen, Geert Braems, Kris Gevaert, Pieter Mestdagh, Jo Vandesompele, Hannelore Denys, Olivier De Wever, An Hendrix Journal J Extracell Vesicles Abstract Separating extracellular vesicles (EV) from blood plasma is challenging and complicates their biolog (show more...)Separating extracellular vesicles (EV) from blood plasma is challenging and complicates their biological understanding and biomarker development. In this study, we fractionate blood plasma by combining size-exclusion chromatography (SEC) and OptiPrep density gradient centrifugation to study clinical context-dependent and time-dependent variations in the biomolecular landscape of systemically circulating EV. Using pooled blood plasma samples from breast cancer patients, we first demonstrate the technical repeatability of blood plasma fractionation. Using serial blood plasma samples from HIV and ovarian cancer patients (n = 10) we next show that EV carry a clinical context-dependent and/or time-dependent protein and small RNA composition, including miRNA and tRNA. In addition, differential analysis of blood plasma fractions provides a catalogue of putative proteins not associated with systemically circulating EV. In conclusion, the implementation of blood plasma fractionation allows to advance the biological understanding and biomarker development of systemically circulating EV. (hide) EV-METRIC 50% (83rd 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. For the quantitative method, the reporting of measured EV concentration is expected. 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 Blood plasma Sample origin ovarian cancer Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: None Proteomics yes EV density (g/ml) 1.09-1.10 Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Proteomics database Yes: Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV200081	4/4	Homo sapiens	Blood plasma	DG SEC (non-commercial)	Vergauwen, Glenn	2021	50%
Study summary Full title Robust sequential biophysical fractionation of blood plasma to study variations in the biomolecular landscape of systemically circulating extracellular vesicles across clinical conditions All authors Glenn Vergauwen, Joeri Tulkens, Cláudio Pinheiro, Francisco Avila Cobos, Sándor Dedeyne, Marie-Angélique De Scheerder, Linos Vandekerckhove, Francis Impens, Ilkka Miinalainen, Geert Braems, Kris Gevaert, Pieter Mestdagh, Jo Vandesompele, Hannelore Denys, Olivier De Wever, An Hendrix Journal J Extracell Vesicles Abstract Separating extracellular vesicles (EV) from blood plasma is challenging and complicates their biolog (show more...)Separating extracellular vesicles (EV) from blood plasma is challenging and complicates their biological understanding and biomarker development. In this study, we fractionate blood plasma by combining size-exclusion chromatography (SEC) and OptiPrep density gradient centrifugation to study clinical context-dependent and time-dependent variations in the biomolecular landscape of systemically circulating EV. Using pooled blood plasma samples from breast cancer patients, we first demonstrate the technical repeatability of blood plasma fractionation. Using serial blood plasma samples from HIV and ovarian cancer patients (n = 10) we next show that EV carry a clinical context-dependent and/or time-dependent protein and small RNA composition, including miRNA and tRNA. In addition, differential analysis of blood plasma fractions provides a catalogue of putative proteins not associated with systemically circulating EV. In conclusion, the implementation of blood plasma fractionation allows to advance the biological understanding and biomarker development of systemically circulating EV. (hide) EV-METRIC 50% (83rd 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. For the quantitative method, the reporting of measured EV concentration is expected. 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 Blood plasma Sample origin HIV Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: None Proteomics yes EV density (g/ml) 1.09-1.10 Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Proteomics database Yes: Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV200081
species	Homo sapiens
sample type	Blood plasma
condition	Control condition	breast cancer	ovarian cancer	HIV
separation protocol	Density gradient Size-exclusion chromatography (non-commercial)	Density gradient Size-exclusion chromatography (non-commercial)	Density gradient Size-exclusion chromatography (non-commercial)	Density gradient Size-exclusion chromatography (non-commercial)
Exp. nr.	1	2	3	4
EV-METRIC %	88	50	50	50