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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV240025	1/5	Homo sapiens	Blood plasma	DC UF Asymmetric-flow field-flow fractionation	Hu L	2024	75%
Study summary Full title Optimized AF4 combined with density cushion ultracentrifugation enables profiling of high-purity human blood extracellular vesicles. All authors Hu L, Zheng X, Zhou M, Wang J, Tong L, Dong M, Xu T, Li Z Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) have emerged as a promising tool for clinical liquid biopsy. However, t (show more...)Extracellular vesicles (EVs) have emerged as a promising tool for clinical liquid biopsy. However, the identification of EVs derived from blood samples is hindered by the presence of abundant plasma proteins, which impairs the downstream biochemical analysis of EV-associated proteins and nucleic acids. Here, we employed optimized asymmetric flow field-flow fractionation (AF4) combined with density cushion ultracentrifugation (UC) to obtain high-purity and intact EVs with very low lipoprotein contamination from human plasma and serum. Further proteomic analysis revealed more than 1000 EV-associated proteins, a large proportion of which has not been previously reported. Specifically, we found that cell-line-derived EV markers are incompatible with the identification of plasma-EVs and proposed that the proteins MYCT1, TSPAN14, MPIG6B and MYADM, as well as the traditional EV markers CD63 and CD147, are plasma-EV markers. Benefiting from the high-purity of EVs, we conducted comprehensive miRNA profiling of plasma EVs and nanosized particles (NPs), as well as compared plasma- and serum-derived EVs, which provides a valuable resource for the EV research community. Overall, our findings provide a comprehensive assessment of human blood EVs as a basis for clinical biopsy applications. (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 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 cushion Ultrafiltration Asymmetric-flow field-flow fractionation Protein markers EV: Alix/ CD9/ Syntenin-1/ CD147/ TSPAN14/ MYCT1 non-EV: Albumin/ ApoA1/ ApoB Proteomics yes Show all info Study aim Biomarker/New methodological development Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 50 Membrane type Regenerated cellulose Density cushion Density medium glycerol Sample volume 1.25 Cushion volume 0.25 Density of the cushion 20% Centrifugation time 90 Centrifugation speed 150000 Other Name other separation method Asymmetric-flow field-flow fractionation Other Name other separation method Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Alix/ CD9/ Syntenin-1/ CD147/ TSPAN14/ MYCT1 Detected contaminants ApoB Not detected contaminants Albumin/ ApoA1 Proteomics database ProteomeXchange Characterization: RNA analysis RNA analysis Type RNA-sequencing Database Genome Sequence Archive Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 132 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV240025	3/5	Homo sapiens	Blood plasma	(d)(U)C	Hu L	2024	63%
Study summary Full title Optimized AF4 combined with density cushion ultracentrifugation enables profiling of high-purity human blood extracellular vesicles. All authors Hu L, Zheng X, Zhou M, Wang J, Tong L, Dong M, Xu T, Li Z Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) have emerged as a promising tool for clinical liquid biopsy. However, t (show more...)Extracellular vesicles (EVs) have emerged as a promising tool for clinical liquid biopsy. However, the identification of EVs derived from blood samples is hindered by the presence of abundant plasma proteins, which impairs the downstream biochemical analysis of EV-associated proteins and nucleic acids. Here, we employed optimized asymmetric flow field-flow fractionation (AF4) combined with density cushion ultracentrifugation (UC) to obtain high-purity and intact EVs with very low lipoprotein contamination from human plasma and serum. Further proteomic analysis revealed more than 1000 EV-associated proteins, a large proportion of which has not been previously reported. Specifically, we found that cell-line-derived EV markers are incompatible with the identification of plasma-EVs and proposed that the proteins MYCT1, TSPAN14, MPIG6B and MYADM, as well as the traditional EV markers CD63 and CD147, are plasma-EV markers. Benefiting from the high-purity of EVs, we conducted comprehensive miRNA profiling of plasma EVs and nanosized particles (NPs), as well as compared plasma- and serum-derived EVs, which provides a valuable resource for the EV research community. Overall, our findings provide a comprehensive assessment of human blood EVs as a basis for clinical biopsy applications. (hide) EV-METRIC 63% (90th 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 (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD9/ Syntenin-1 non-EV: Albumin/ ApoA1/ ApoB Proteomics no Show all info Study aim Biomarker/New methodological development Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Equal to or above 150,000 g Pelleting performed Yes Pelleting: rotor type TLS-55 Pelleting: speed (g) 150000 Other Name other separation method Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Alix/ CD9/ Syntenin-1 Detected contaminants Albumin/ ApoA1/ ApoB Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field

	EV240025	4/5	Homo sapiens	Blood plasma	DC	Hu L	2024	63%
Study summary Full title Optimized AF4 combined with density cushion ultracentrifugation enables profiling of high-purity human blood extracellular vesicles. All authors Hu L, Zheng X, Zhou M, Wang J, Tong L, Dong M, Xu T, Li Z Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) have emerged as a promising tool for clinical liquid biopsy. However, t (show more...)Extracellular vesicles (EVs) have emerged as a promising tool for clinical liquid biopsy. However, the identification of EVs derived from blood samples is hindered by the presence of abundant plasma proteins, which impairs the downstream biochemical analysis of EV-associated proteins and nucleic acids. Here, we employed optimized asymmetric flow field-flow fractionation (AF4) combined with density cushion ultracentrifugation (UC) to obtain high-purity and intact EVs with very low lipoprotein contamination from human plasma and serum. Further proteomic analysis revealed more than 1000 EV-associated proteins, a large proportion of which has not been previously reported. Specifically, we found that cell-line-derived EV markers are incompatible with the identification of plasma-EVs and proposed that the proteins MYCT1, TSPAN14, MPIG6B and MYADM, as well as the traditional EV markers CD63 and CD147, are plasma-EV markers. Benefiting from the high-purity of EVs, we conducted comprehensive miRNA profiling of plasma EVs and nanosized particles (NPs), as well as compared plasma- and serum-derived EVs, which provides a valuable resource for the EV research community. Overall, our findings provide a comprehensive assessment of human blood EVs as a basis for clinical biopsy applications. (hide) EV-METRIC 63% (90th 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 cushion Protein markers EV: Alix/ CD9/ Syntenin-1 non-EV: Albumin/ ApoA1/ ApoB Proteomics no Show all info Study aim Biomarker/New methodological development Sample Species Homo sapiens Sample Type Blood plasma Separation Method Density cushion Density medium glycerol Sample volume 1.25 Cushion volume 0.25 Density of the cushion 20% Centrifugation time 90 Centrifugation speed 150000 Other Name other separation method Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Alix/ CD9/ Syntenin-1 Detected contaminants Albumin/ ApoA1/ ApoB Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 105.1 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.85E+10 EM EM-type Transmission-EM Image type Wide-field

	EV240025	5/5	Homo sapiens	Blood plasma	DC qEV35	Hu L	2024	63%
Study summary Full title Optimized AF4 combined with density cushion ultracentrifugation enables profiling of high-purity human blood extracellular vesicles. All authors Hu L, Zheng X, Zhou M, Wang J, Tong L, Dong M, Xu T, Li Z Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) have emerged as a promising tool for clinical liquid biopsy. However, t (show more...)Extracellular vesicles (EVs) have emerged as a promising tool for clinical liquid biopsy. However, the identification of EVs derived from blood samples is hindered by the presence of abundant plasma proteins, which impairs the downstream biochemical analysis of EV-associated proteins and nucleic acids. Here, we employed optimized asymmetric flow field-flow fractionation (AF4) combined with density cushion ultracentrifugation (UC) to obtain high-purity and intact EVs with very low lipoprotein contamination from human plasma and serum. Further proteomic analysis revealed more than 1000 EV-associated proteins, a large proportion of which has not been previously reported. Specifically, we found that cell-line-derived EV markers are incompatible with the identification of plasma-EVs and proposed that the proteins MYCT1, TSPAN14, MPIG6B and MYADM, as well as the traditional EV markers CD63 and CD147, are plasma-EV markers. Benefiting from the high-purity of EVs, we conducted comprehensive miRNA profiling of plasma EVs and nanosized particles (NPs), as well as compared plasma- and serum-derived EVs, which provides a valuable resource for the EV research community. Overall, our findings provide a comprehensive assessment of human blood EVs as a basis for clinical biopsy applications. (hide) EV-METRIC 63% (90th 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 cushion Commercial method Protein markers EV: Alix/ CD9/ Syntenin-1 non-EV: Albumin/ ApoA1/ ApoB Proteomics no Show all info Study aim Biomarker/New methodological development Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV35 Density cushion Density medium glycerol Sample volume 1.25 Cushion volume 0.25 Density of the cushion 20% Centrifugation time 90 Centrifugation speed 150000 Other Name other separation method Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Alix/ CD9/ Syntenin-1 Detected contaminants Albumin/ ApoA1/ ApoB Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 103.8 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.80E+09 EM EM-type Transmission-EM Image type Wide-field

	EV240025	2/5	Homo sapiens	Serum	DC UF Asymmetric-flow field-flow fractionation	Hu L	2024	33%
Study summary Full title Optimized AF4 combined with density cushion ultracentrifugation enables profiling of high-purity human blood extracellular vesicles. All authors Hu L, Zheng X, Zhou M, Wang J, Tong L, Dong M, Xu T, Li Z Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) have emerged as a promising tool for clinical liquid biopsy. However, t (show more...)Extracellular vesicles (EVs) have emerged as a promising tool for clinical liquid biopsy. However, the identification of EVs derived from blood samples is hindered by the presence of abundant plasma proteins, which impairs the downstream biochemical analysis of EV-associated proteins and nucleic acids. Here, we employed optimized asymmetric flow field-flow fractionation (AF4) combined with density cushion ultracentrifugation (UC) to obtain high-purity and intact EVs with very low lipoprotein contamination from human plasma and serum. Further proteomic analysis revealed more than 1000 EV-associated proteins, a large proportion of which has not been previously reported. Specifically, we found that cell-line-derived EV markers are incompatible with the identification of plasma-EVs and proposed that the proteins MYCT1, TSPAN14, MPIG6B and MYADM, as well as the traditional EV markers CD63 and CD147, are plasma-EV markers. Benefiting from the high-purity of EVs, we conducted comprehensive miRNA profiling of plasma EVs and nanosized particles (NPs), as well as compared plasma- and serum-derived EVs, which provides a valuable resource for the EV research community. Overall, our findings provide a comprehensive assessment of human blood EVs as a basis for clinical biopsy applications. (hide) EV-METRIC 33% (76th 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 Serum 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 cushion Ultrafiltration Asymmetric-flow field-flow fractionation Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biomarker/New methodological development Sample Species Homo sapiens Sample Type Serum Separation Method Ultra filtration Cut-off size (kDa) 50 Membrane type Regenerated cellulose Density cushion Density medium glycerol Sample volume 1.25 Cushion volume 0.25 Density of the cushion 20% Centrifugation time 90 Centrifugation speed 150000 Other Name other separation method Asymmetric-flow field-flow fractionation Other Name other separation method Characterization: Protein analysis Protein Concentration Method BCA Proteomics database ProteomeXchange Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV240025
species	Homo sapiens
sample type	Blood plasma	Blood plasma	Blood plasma	Blood plasma	Serum
condition	Control condition	Control condition	Control condition	Control condition	Control condition
separation protocol	DC/ Ultrafiltration/ Asymmetric-flow field-flow fractionation	dUC	DC	DC/ qEV35	DC/ Ultrafiltration/ Asymmetric-flow field-flow fractionation
Exp. nr.	1	3	4	5	2
EV-METRIC %	75	63	63	63	33