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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV100012	1/2	Mus musculus	Urine	(d)(U)C DC Filtration	Conde-Vancells J	2010	33%
Study summary Full title Candidate biomarkers in exosome-like vesicles purified from rat and mouse urine samples All authors Conde-Vancells J, Rodriguez-Suarez E, Gonzalez E, Berisa A, Gil D, Embade N, Valle M, Luka Z, Elortza F, Wagner C, Lu SC, Mato JM, Falcon-Perez M Journal Proteomics Clin Appl Abstract PURPOSE: There is a compelling clinical imperative to identify discerning molecular biomarkers of he (show more...)PURPOSE: There is a compelling clinical imperative to identify discerning molecular biomarkers of hepatic disease in order to inform the diagnosis, prognosis and treatment. EXPERIMENTAL DESIGN: We have investigated the proteome of urinary vesicles present in urine samples obtained from experimental models for the study of liver injury, as an approach for identifying potential biomarkers for hepatic disease. RESULTS: The biochemical and proteomic characterization of highly purified exosome-like urinary vesicles has identified 28 proteins previously unreported in these vesicles, and many that have been previously associated with diseases, such as the prion-related protein. Furthermore, in urine samples from D-galactosamine-treated rats, a well-characterized experimental model for acute liver injury, we have detected a severe reduction in some proteins that normally are clearly detected in urinary vesicles. Finally, differential protein content on urinary vesicles from a mouse model for chronic liver injury has been also identified. CONCLUSIONS AND CLINICAL RELEVANCE: Our results argue positively that urinary vesicles could be a source for identifying non-invasive biomarkers of liver injury. We proposed some proteins such as Cd26, Cd81, Slc3A1 and Cd10 that have been found to be differentially expressed in urinary vesicles from some of the analyzed models as potential biomarkers for liver injury. (hide) EV-METRIC 33% (65th 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 Urine Sample origin NAY Focus vesicles Exosome-like vesicles 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 (d)(U)C DC Filtration Protein markers EV: TSG101/ CD63/ LimpII/ AQP1/ CD81/ Slc3a1/ Flotillin/ CD26/ PrP/ CD10/ HSP70 non-EV: Tamm-Horsfall glycoprotein Proteomics yes TEM measurements 95.8+-50.7 Show all info Study aim Biomarker Sample Species Mus musculus Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ HSP70/ TSG101/ CD26/ CD10/ LimpII/ PrP/ Slc3a1/ AQP1/ Flotillin Detected contaminants Tamm-Horsfall glycoprotein ELISA Antibody details provided? No Detected EV-associated proteins CD26/ CD10/ LimpII/ PrP/ Slc3a1/ AQP1/ Flotillin Characterization: Particle analysis EM EM-type cryo EM Image type Close-up Report size (nm) 95.8+-50.7

	EV100012	2/2	Rattus norvegicus/rattus	Urine	(d)(U)C DG Filtration	Conde-Vancells J	2010	33%
Study summary Full title Candidate biomarkers in exosome-like vesicles purified from rat and mouse urine samples All authors Conde-Vancells J, Rodriguez-Suarez E, Gonzalez E, Berisa A, Gil D, Embade N, Valle M, Luka Z, Elortza F, Wagner C, Lu SC, Mato JM, Falcon-Perez M Journal Proteomics Clin Appl Abstract PURPOSE: There is a compelling clinical imperative to identify discerning molecular biomarkers of he (show more...)PURPOSE: There is a compelling clinical imperative to identify discerning molecular biomarkers of hepatic disease in order to inform the diagnosis, prognosis and treatment. EXPERIMENTAL DESIGN: We have investigated the proteome of urinary vesicles present in urine samples obtained from experimental models for the study of liver injury, as an approach for identifying potential biomarkers for hepatic disease. RESULTS: The biochemical and proteomic characterization of highly purified exosome-like urinary vesicles has identified 28 proteins previously unreported in these vesicles, and many that have been previously associated with diseases, such as the prion-related protein. Furthermore, in urine samples from D-galactosamine-treated rats, a well-characterized experimental model for acute liver injury, we have detected a severe reduction in some proteins that normally are clearly detected in urinary vesicles. Finally, differential protein content on urinary vesicles from a mouse model for chronic liver injury has been also identified. CONCLUSIONS AND CLINICAL RELEVANCE: Our results argue positively that urinary vesicles could be a source for identifying non-invasive biomarkers of liver injury. We proposed some proteins such as Cd26, Cd81, Slc3A1 and Cd10 that have been found to be differentially expressed in urinary vesicles from some of the analyzed models as potential biomarkers for liver injury. (hide) EV-METRIC 33% (65th 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 Urine Sample origin NAY Focus vesicles Exosome-like vesicles 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 (d)(U)C DG Filtration Protein markers EV: TSG101/ Caveolin/ CD63/ CD81/ Slc3a1/ Flotillin/ CD10 non-EV: Proteomics no EV density (g/ml) 1.18;1.246;1.252 Show all info Study aim Biomarker Sample Species Rattus norvegicus/rattus Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Bottom-up Rotor type TLA110 Speed (g) 210000 Pelleting-wash: volume per pellet (mL) 2 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ TSG101/ CD10/ Slc3a1/ Caveolin/ Flotillin ELISA Antibody details provided? No Detected EV-associated proteins CD10/ Slc3a1/ Caveolin/ Flotillin Characterization: Particle analysis EM EM-type cryo EM Image type Wide-field

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EV-TRACK ID	EV100012
species	Mus musculus	Rattus norvegicus/rattus
sample type	Urine	Urine
condition	NAY	NAY
separation protocol	(d)(U)C DC Filtration	(d)(U)C DG Filtration
Exp. nr.	1	2
EV-METRIC %	33	33