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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210036	1/2	Homo sapiens	HT-29	(d)(U)C UF	Knol, Jaco C	2016	29%
Study summary Full title Peptide-mediated 'miniprep' isolation of extracellular vesicles is suitable for high-throughput proteomics All authors Jaco C Knol, Inge de Reus, Tim Schelfhorst, Robin Beekhof, Meike de Wit, Sander R Piersma, Thang V Pham, Egbert F Smit, Henk M W Verheul, Connie R Jiménez Journal EuPA Open Proteom. Abstract Extracellular vesicles (EVs) are cell-secreted membrane vesicles enclosed by a lipid bilayer derived (show more...)Extracellular vesicles (EVs) are cell-secreted membrane vesicles enclosed by a lipid bilayer derived from endosomes or from the plasma membrane. Since EVs are released into body fluids, and their cargo includes tissue-specific and disease-related molecules, they represent a rich source for disease biomarkers. However, standard ultracentrifugation methods for EV isolation are laborious, time-consuming, and require high inputs. Ghosh and co-workers recently described an isolation method utilizing Heat Shock Protein (HSP)-binding peptide Vn96 to aggregate HSP-decorated EVs, which can be performed at small 'miniprep' scale. Based on microscopic, immunoblot, and RNA sequencing analyses this method compared well with ultracentrifugation-mediated EV isolation, but a detailed proteomic comparison was lacking. Therefore, we compared both methods using label-free proteomics of replicate EV isolations from HT-29 cell-conditioned medium. Despite a 30-fold different scale (ultracentrifugation: 60 ml/Vn96-mediated aggregation: 2 ml) both methods yielded comparable numbers of identified proteins (3115/3085), with similar reproducibility of identification (72.5%/75.5%) and spectral count-based quantification (average CV: 31%/27%). EV fractions obtained with either method contained established EV markers and proteins linked to vesicle-related gene ontologies. Thus, Vn96 peptide-mediated aggregation is an advantageous, simple and rapid approach for EV isolation from small biological samples, enabling high-throughput analysis in a biomarker discovery setting. (hide) EV-METRIC 29% (68th 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 (Differential) (ultra)centrifugation Ultrafiltration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HT-29 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 114000 Wash: volume per pellet (ml) 13.2 Wash: time (min) 90 Wash: Rotor Type SW 40 Ti Wash: speed (g) 114000 Ultra filtration Cut-off size (kDa) 3 Membrane type Not specified Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database No Characterization: Lipid analysis No

	EV210036	2/2	Homo sapiens	HT-29	UF ME kit (New England Peptide)	Knol, Jaco C	2016	17%
Study summary Full title Peptide-mediated 'miniprep' isolation of extracellular vesicles is suitable for high-throughput proteomics All authors Jaco C Knol, Inge de Reus, Tim Schelfhorst, Robin Beekhof, Meike de Wit, Sander R Piersma, Thang V Pham, Egbert F Smit, Henk M W Verheul, Connie R Jiménez Journal EuPA Open Proteom. Abstract Extracellular vesicles (EVs) are cell-secreted membrane vesicles enclosed by a lipid bilayer derived (show more...)Extracellular vesicles (EVs) are cell-secreted membrane vesicles enclosed by a lipid bilayer derived from endosomes or from the plasma membrane. Since EVs are released into body fluids, and their cargo includes tissue-specific and disease-related molecules, they represent a rich source for disease biomarkers. However, standard ultracentrifugation methods for EV isolation are laborious, time-consuming, and require high inputs. Ghosh and co-workers recently described an isolation method utilizing Heat Shock Protein (HSP)-binding peptide Vn96 to aggregate HSP-decorated EVs, which can be performed at small 'miniprep' scale. Based on microscopic, immunoblot, and RNA sequencing analyses this method compared well with ultracentrifugation-mediated EV isolation, but a detailed proteomic comparison was lacking. Therefore, we compared both methods using label-free proteomics of replicate EV isolations from HT-29 cell-conditioned medium. Despite a 30-fold different scale (ultracentrifugation: 60 ml/Vn96-mediated aggregation: 2 ml) both methods yielded comparable numbers of identified proteins (3115/3085), with similar reproducibility of identification (72.5%/75.5%) and spectral count-based quantification (average CV: 31%/27%). EV fractions obtained with either method contained established EV markers and proteins linked to vesicle-related gene ontologies. Thus, Vn96 peptide-mediated aggregation is an advantageous, simple and rapid approach for EV isolation from small biological samples, enabling high-throughput analysis in a biomarker discovery setting. (hide) EV-METRIC 17% (54th 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 Ultrafiltration Commercial method Protein markers EV: CD81/ HSP70/ CD63 non-EV: None Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HT-29 EV-harvesting Medium Serum free medium Separation Method Ultra filtration Cut-off size (kDa) 3 Membrane type Not specified Commercial kit ME kit (New England Peptide) Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ HSP70/ CD81 Proteomics database No Characterization: Lipid analysis No

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EV-TRACK ID	EV210036
species	Homo sapiens
sample type	Cell culture
cell type	HT-29
condition	Control condition
separation protocol	dUC Ultrafiltration	Ultrafiltration ME kit (New England Peptide)
Exp. nr.	1	2
EV-METRIC %	29	17