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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200102	1/7	Homo sapiens	THP1	DG (d)(U)C qEV	Tóth, Eszter	2021	100%
Study summary Full title Formation of a protein corona on the surface of extracellular vesicles in blood plasma All authors Eszter Á Tóth, Lilla Turiák, Tamás Visnovitz, Csaba Cserép, Anett Mázló, Barbara W Sódar, András I Försönits, Gábor Petővári, Anna Sebestyén, Zsolt Komlósi, László Drahos, Ágnes Kittel, György Nagy, Attila Bácsi, Ádám Dénes, Yong Song Gho, Katalin É Szabó-Taylor, Edit I Buzás Journal J Extracell Vesicles Abstract In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in bl (show more...)In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium-sized nascent EVs of THP1 cells as well as of Optiprep-purified platelets, and incubated them in EV-depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein-coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α-chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF-α, IL-6, CD83, CD86 and HLA-DR of human monocyte-derived dendritic cells, EV-free protein aggregates had no effect. In conclusion, our data may shed new light on the origin of the commonly reported plasma protein 'contamination' of EV preparations and may add a new perspective to EV research. (hide) EV-METRIC 100% (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 Cell culture supernatant 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 (Differential) (ultra)centrifugation Commercial method Protein markers EV: CD63/ Phosphatydilserine non-EV: None Proteomics yes EV density (g/ml) 1.10-1.15 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells THP1 EV-harvesting Medium Serum free medium Cell viability (%) 93 Cell count 80000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 40 Pelleting: rotor type FA-45-24-11 Pelleting: speed (g) 12500 Wash: volume per pellet (ml) 1 Wash: time (min) 40 Wash: Rotor Type FA-45-24-11 Wash: speed (g) 12500 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 4.5 Sample volume (mL) 0.5 Orientation Top-down Rotor type MLS-50 Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 2 Pelleting: duration (min) 80 Pelleting: rotor type FA-45-24-11 Pelleting: speed (g) 12500 Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Flow cytometry Type of Flow cytometry FACS Calibur Calibration bead size The vesicular gate was set using Megamix Beads (Bi Detected EV-associated proteins Phosphatydilserine Proteomics database Yes: Detected EV-associated proteins CD63 Characterization: Lipid analysis Yes Characterization: Particle analysis TRPS Report type Modus Reported size (nm) 244 Particle analysis: flow cytometry Flow cytometer type FACS Calibur Hardware adjustment Calibration bead size 0.160;0.200;0.240;0.500 Report type Not Reported EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200102	4/7	Homo sapiens	Blood plasma	DG	Tóth, Eszter	2021	86%
Study summary Full title Formation of a protein corona on the surface of extracellular vesicles in blood plasma All authors Eszter Á Tóth, Lilla Turiák, Tamás Visnovitz, Csaba Cserép, Anett Mázló, Barbara W Sódar, András I Försönits, Gábor Petővári, Anna Sebestyén, Zsolt Komlósi, László Drahos, Ágnes Kittel, György Nagy, Attila Bácsi, Ádám Dénes, Yong Song Gho, Katalin É Szabó-Taylor, Edit I Buzás Journal J Extracell Vesicles Abstract In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in bl (show more...)In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium-sized nascent EVs of THP1 cells as well as of Optiprep-purified platelets, and incubated them in EV-depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein-coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α-chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF-α, IL-6, CD83, CD86 and HLA-DR of human monocyte-derived dendritic cells, EV-free protein aggregates had no effect. In conclusion, our data may shed new light on the origin of the commonly reported plasma protein 'contamination' of EV preparations and may add a new perspective to EV research. (hide) EV-METRIC 86% (98th 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: EV-depleted plasma, spiked with THP1 EVs 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 Protein markers EV: CD63/ Phosphatydilserine non-EV: None Proteomics yes EV density (g/ml) 1.10-1.15 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 4.5 Sample volume (mL) 0.5 Orientation Top-down Rotor type MLS-50 Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 2 Pelleting: duration (min) 80 Pelleting: rotor type FA-45-24-11 Pelleting: speed (g) 12500 Characterization: Protein analysis Protein Concentration Method microBCA Flow cytometry Type of Flow cytometry FACS Calibur Calibration bead size The vesicular gate was set using Megamix Beads (Bi Detected EV-associated proteins Phosphatydilserine Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type FACS Calibur Hardware adjustment Calibration bead size 0.160;0.200;0.240;0.500 Report type Not Reported EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200102	2/7	Homo sapiens	Blood plasma	(d)(U)C	Tóth, Eszter	2021	75%
Study summary Full title Formation of a protein corona on the surface of extracellular vesicles in blood plasma All authors Eszter Á Tóth, Lilla Turiák, Tamás Visnovitz, Csaba Cserép, Anett Mázló, Barbara W Sódar, András I Försönits, Gábor Petővári, Anna Sebestyén, Zsolt Komlósi, László Drahos, Ágnes Kittel, György Nagy, Attila Bácsi, Ádám Dénes, Yong Song Gho, Katalin É Szabó-Taylor, Edit I Buzás Journal J Extracell Vesicles Abstract In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in bl (show more...)In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium-sized nascent EVs of THP1 cells as well as of Optiprep-purified platelets, and incubated them in EV-depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein-coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α-chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF-α, IL-6, CD83, CD86 and HLA-DR of human monocyte-derived dendritic cells, EV-free protein aggregates had no effect. In conclusion, our data may shed new light on the origin of the commonly reported plasma protein 'contamination' of EV preparations and may add a new perspective to EV research. (hide) EV-METRIC 75% (96th 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: EV-depleted plasma, spiked with THP1 EVs 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 (Differential) (ultra)centrifugation Protein markers EV: CD63/ Phosphatydilserine non-EV: Albumin/ fibrinogen/ haptoglobin/ complement C3 Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 40 Pelleting: rotor type FA-45-24-11 Pelleting: speed (g) 12500 Wash: volume per pellet (ml) 1 Wash: time (min) 40 Wash: Rotor Type FA-45-24-11 Wash: speed (g) 12500 Characterization: Protein analysis Protein Concentration Method microBCA Flow cytometry Type of Flow cytometry FACS Calibur Calibration bead size The vesicular gate was set using Megamix Beads (Bi Detected EV-associated proteins Phosphatydilserine Proteomics database Yes: Detected EV-associated proteins CD63 Detected contaminants fibrinogen/ haptoglobin/ complement C3/ Albumin Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Modus Reported size (nm) 240 Particle analysis: flow cytometry Flow cytometer type FACS Calibur Hardware adjustment Calibration bead size 0.160;0.200;0.240;0.500 Report type Not Reported EM EM-type Immuno-EM/ Transmission-EM EM protein Other;CD63, plasma proteins Image type Close-up, Wide-field

	EV200102	5/7	Homo sapiens	Blood plasma	(d)(U)C	Tóth, Eszter	2021	75%
Study summary Full title Formation of a protein corona on the surface of extracellular vesicles in blood plasma All authors Eszter Á Tóth, Lilla Turiák, Tamás Visnovitz, Csaba Cserép, Anett Mázló, Barbara W Sódar, András I Försönits, Gábor Petővári, Anna Sebestyén, Zsolt Komlósi, László Drahos, Ágnes Kittel, György Nagy, Attila Bácsi, Ádám Dénes, Yong Song Gho, Katalin É Szabó-Taylor, Edit I Buzás Journal J Extracell Vesicles Abstract In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in bl (show more...)In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium-sized nascent EVs of THP1 cells as well as of Optiprep-purified platelets, and incubated them in EV-depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein-coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α-chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF-α, IL-6, CD83, CD86 and HLA-DR of human monocyte-derived dendritic cells, EV-free protein aggregates had no effect. In conclusion, our data may shed new light on the origin of the commonly reported plasma protein 'contamination' of EV preparations and may add a new perspective to EV research. (hide) EV-METRIC 75% (96th 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 Rheuma: EV-depleted plasma, spiked with THP1 EVs 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 (Differential) (ultra)centrifugation Protein markers EV: CD63/ Phosphatydilserine non-EV: Albumin/ fibrinogen/ haptoglobin/ complement C3 Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 40 Pelleting: rotor type FA-45-24-11 Pelleting: speed (g) 12500 Wash: volume per pellet (ml) 1 Wash: time (min) 40 Wash: Rotor Type FA-45-24-11 Wash: speed (g) 12500 Characterization: Protein analysis Protein Concentration Method microBCA Flow cytometry Type of Flow cytometry FACS Calibur Calibration bead size The vesicular gate was set using Megamix Beads (Bi Detected EV-associated proteins Phosphatydilserine Proteomics database Yes: Detected EV-associated proteins CD63 Detected contaminants fibrinogen/ haptoglobin/ complement C3/ Albumin Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Not Reported Particle analysis: flow cytometry Flow cytometer type FACS Calibur Hardware adjustment Calibration bead size 0.160;0.200;0.240;0.500 Report type Not Reported

	EV200102	7/7	Homo sapiens	Blood plasma	DG	Tóth, Eszter	2021	71%
Study summary Full title Formation of a protein corona on the surface of extracellular vesicles in blood plasma All authors Eszter Á Tóth, Lilla Turiák, Tamás Visnovitz, Csaba Cserép, Anett Mázló, Barbara W Sódar, András I Försönits, Gábor Petővári, Anna Sebestyén, Zsolt Komlósi, László Drahos, Ágnes Kittel, György Nagy, Attila Bácsi, Ádám Dénes, Yong Song Gho, Katalin É Szabó-Taylor, Edit I Buzás Journal J Extracell Vesicles Abstract In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in bl (show more...)In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium-sized nascent EVs of THP1 cells as well as of Optiprep-purified platelets, and incubated them in EV-depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein-coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α-chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF-α, IL-6, CD83, CD86 and HLA-DR of human monocyte-derived dendritic cells, EV-free protein aggregates had no effect. In conclusion, our data may shed new light on the origin of the commonly reported plasma protein 'contamination' of EV preparations and may add a new perspective to EV research. (hide) EV-METRIC 71% (94th 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 Rheuma: EV-depleted plasma, spiked with THP1 EVs 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 Protein markers EV: CD63/ Phosphatydilserine non-EV: None Proteomics yes EV density (g/ml) 1.10-1.15 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 4.5 Sample volume (mL) 0.5 Orientation Top-down Rotor type MLS-50 Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 2 Pelleting: duration (min) 80 Pelleting: rotor type FA-45-24-11 Pelleting: speed (g) 12500 Characterization: Protein analysis Protein Concentration Method microBCA Flow cytometry Type of Flow cytometry FACS Calibur Calibration bead size The vesicular gate was set using Megamix Beads (Bi Detected EV-associated proteins Phosphatydilserine Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type FACS Calibur Hardware adjustment Calibration bead size 0.160;0.200;0.240;0.500 Report type Not Reported

	EV200102	3/7	Homo sapiens	Blood plasma	qEV	Tóth, Eszter	2021	57%
Study summary Full title Formation of a protein corona on the surface of extracellular vesicles in blood plasma All authors Eszter Á Tóth, Lilla Turiák, Tamás Visnovitz, Csaba Cserép, Anett Mázló, Barbara W Sódar, András I Försönits, Gábor Petővári, Anna Sebestyén, Zsolt Komlósi, László Drahos, Ágnes Kittel, György Nagy, Attila Bácsi, Ádám Dénes, Yong Song Gho, Katalin É Szabó-Taylor, Edit I Buzás Journal J Extracell Vesicles Abstract In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in bl (show more...)In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium-sized nascent EVs of THP1 cells as well as of Optiprep-purified platelets, and incubated them in EV-depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein-coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α-chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF-α, IL-6, CD83, CD86 and HLA-DR of human monocyte-derived dendritic cells, EV-free protein aggregates had no effect. In conclusion, our data may shed new light on the origin of the commonly reported plasma protein 'contamination' of EV preparations and may add a new perspective to EV research. (hide) EV-METRIC 57% (89th 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: EV-depleted plasma, spiked with THP1 EVs 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 qEV Protein markers EV: CD63/ Phosphatydilserine non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method microBCA Flow cytometry Type of Flow cytometry FACS Calibur Calibration bead size The vesicular gate was set using Megamix Beads (Bi Detected EV-associated proteins Phosphatydilserine Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type FACS Calibur Hardware adjustment Calibration bead size 0.160;0.200;0.240;0.500 Report type Not Reported EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200102	6/7	Homo sapiens	Blood plasma	qEV	Tóth, Eszter	2021	43%
Study summary Full title Formation of a protein corona on the surface of extracellular vesicles in blood plasma All authors Eszter Á Tóth, Lilla Turiák, Tamás Visnovitz, Csaba Cserép, Anett Mázló, Barbara W Sódar, András I Försönits, Gábor Petővári, Anna Sebestyén, Zsolt Komlósi, László Drahos, Ágnes Kittel, György Nagy, Attila Bácsi, Ádám Dénes, Yong Song Gho, Katalin É Szabó-Taylor, Edit I Buzás Journal J Extracell Vesicles Abstract In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in bl (show more...)In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium-sized nascent EVs of THP1 cells as well as of Optiprep-purified platelets, and incubated them in EV-depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein-coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α-chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF-α, IL-6, CD83, CD86 and HLA-DR of human monocyte-derived dendritic cells, EV-free protein aggregates had no effect. In conclusion, our data may shed new light on the origin of the commonly reported plasma protein 'contamination' of EV preparations and may add a new perspective to EV research. (hide) EV-METRIC 43% (74th 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 Rheuma: EV-depleted plasma, spiked with THP1 EVs 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 qEV Protein markers EV: CD63/ Phosphatydilserine non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method microBCA Flow cytometry Type of Flow cytometry FACS Calibur Calibration bead size The vesicular gate was set using Megamix Beads (Bi Detected EV-associated proteins Phosphatydilserine Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type FACS Calibur Hardware adjustment Calibration bead size 0.160;0.200;0.240;0.500 Report type Not Reported

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EV-TRACK ID	EV200102
species	Homo sapiens
sample type	Cell culture	Blood plasma	Blood plasma	Blood plasma	Blood plasma	Blood plasma	Blood plasma
cell type	THP1	NA	NA	NA	NA	NA	NA
medium	Serum free medium	NA	NA	NA	NA	NA	NA
condition	Control condition	Control: EV-depleted plasma spiked with THP1 EVs	Control: EV-depleted plasma spiked with THP1 EVs	Rheuma: EV-depleted plasma spiked with THP1 EVs	Rheuma: EV-depleted plasma spiked with THP1 EVs	Control: EV-depleted plasma spiked with THP1 EVs	Rheuma: EV-depleted plasma spiked with THP1 EVs
separation protocol	Density gradient dUC qEV	Density gradient	dUC	dUC	Density gradient	qEV	qEV
Exp. nr.	1	4	2	5	7	3	6
EV-METRIC %	100	86	75	75	71	57	43

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