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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210188	2/3	Homo sapiens	Urine	(d)(U)C Filtration	Sequeiros, Tamara	2017	33%
Study summary Full title Targeted proteomics in urinary extracellular vesicles identifies biomarkers for diagnosis and prognosis of prostate cancer All authors Tamara Sequeiros, Marina Rigau, Cristina Chiva, Melania Montes, Iolanda Garcia-Grau, Marta Garcia, Sherley Diaz, Ana Celma, Irene Bijnsdorp, Alex Campos, Primiano Di Mauro, Salvador Borrós, Jaume Reventós 10 , Andreas Doll, Rosanna Paciucci, Michiel Pegtel 11 , Inés de Torres, Eduard Sabidó, Juan Morote, Mireia Olivan Journal Oncotarget Abstract Rapid and reliable diagnosis of prostate cancer (PCa) is highly desirable as current used methods la (show more...)Rapid and reliable diagnosis of prostate cancer (PCa) is highly desirable as current used methods lack specificity. In addition, identification of PCa biomarkers that can classify patients into high- and low-risk groups for disease progression at early stage will improve treatment decision-making. Here, we describe a set of protein-combination panels in urinary extracellular vesicles (EVs), defined by targeted proteomics and immunoblotting techniques that improve early non-invasive detection and stratification of PCa patients.We report a two-protein combination in urinary EVs that classifies benign and PCa patients (ADSV-TGM4), and a combination of five proteins able to significantly distinguish between high- and low-grade PCa patients (CD63-GLPK5-SPHM-PSA-PAPP). Proteins composing the panels were validated by immunohistochemistry assays in tissue microarrays (TMAs) confirming a strong link between the urinary EVs proteome and alterations in PCa tissues. Moreover, ADSV and TGM4 abundance yielded a high diagnostic potential in tissue and promising TGM4 prognostic power. These results suggest that the proteins identified in urinary EVs distinguishing high- and low grade PCa are a reflection of histological changes that may be a consequence of their functional involvement in PCa development. In conclusion, our study resulted in the identification of protein-combination panels present in urinary EVs that exhibit high sensitivity and specificity for PCa detection and patient stratification. Moreover, our study highlights the potential of targeted proteomic approaches-such as selected reaction monitoring (SRM)-as diagnostic assay for liquid biopsies via urinary EVs to improve diagnosis and prognosis of suspected PCa patients. (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 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 (d)(U)C Filtration Protein markers EV: TSG101/ TGM4/ ADSV/ CD81 non-EV: None Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens 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 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 60 Wash: Rotor Type Not specified Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins ADSV/ TSG101/ CD81/ TGM4 Proteomics database Yes: ProteomeXchange Consortiu Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes Particle yield Not Reported NA EM EM-type Transmission-EM Image type Close-up

	EV210188	1/3	Homo sapiens	Urine	(d)(U)C Filtration	Sequeiros, Tamara	2017	22%
Study summary Full title Targeted proteomics in urinary extracellular vesicles identifies biomarkers for diagnosis and prognosis of prostate cancer All authors Tamara Sequeiros, Marina Rigau, Cristina Chiva, Melania Montes, Iolanda Garcia-Grau, Marta Garcia, Sherley Diaz, Ana Celma, Irene Bijnsdorp, Alex Campos, Primiano Di Mauro, Salvador Borrós, Jaume Reventós 10 , Andreas Doll, Rosanna Paciucci, Michiel Pegtel 11 , Inés de Torres, Eduard Sabidó, Juan Morote, Mireia Olivan Journal Oncotarget Abstract Rapid and reliable diagnosis of prostate cancer (PCa) is highly desirable as current used methods la (show more...)Rapid and reliable diagnosis of prostate cancer (PCa) is highly desirable as current used methods lack specificity. In addition, identification of PCa biomarkers that can classify patients into high- and low-risk groups for disease progression at early stage will improve treatment decision-making. Here, we describe a set of protein-combination panels in urinary extracellular vesicles (EVs), defined by targeted proteomics and immunoblotting techniques that improve early non-invasive detection and stratification of PCa patients.We report a two-protein combination in urinary EVs that classifies benign and PCa patients (ADSV-TGM4), and a combination of five proteins able to significantly distinguish between high- and low-grade PCa patients (CD63-GLPK5-SPHM-PSA-PAPP). Proteins composing the panels were validated by immunohistochemistry assays in tissue microarrays (TMAs) confirming a strong link between the urinary EVs proteome and alterations in PCa tissues. Moreover, ADSV and TGM4 abundance yielded a high diagnostic potential in tissue and promising TGM4 prognostic power. These results suggest that the proteins identified in urinary EVs distinguishing high- and low grade PCa are a reflection of histological changes that may be a consequence of their functional involvement in PCa development. In conclusion, our study resulted in the identification of protein-combination panels present in urinary EVs that exhibit high sensitivity and specificity for PCa detection and patient stratification. Moreover, our study highlights the potential of targeted proteomic approaches-such as selected reaction monitoring (SRM)-as diagnostic assay for liquid biopsies via urinary EVs to improve diagnosis and prognosis of suspected PCa patients. (hide) EV-METRIC 22% (49th 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 Low grade prostate 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 (d)(U)C Filtration Protein markers EV: SPHM/ TSG101/ KLK3/ CD63/ CD81 non-EV: None Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens 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 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 60 Wash: Rotor Type Not specified Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ SPHM/ KLK3/ TSG101/ CD81 Proteomics database Yes: ProteomeXchange Consortiu Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210188	3/3	Homo sapiens	Urine	(d)(U)C Filtration	Sequeiros, Tamara	2017	22%
Study summary Full title Targeted proteomics in urinary extracellular vesicles identifies biomarkers for diagnosis and prognosis of prostate cancer All authors Tamara Sequeiros, Marina Rigau, Cristina Chiva, Melania Montes, Iolanda Garcia-Grau, Marta Garcia, Sherley Diaz, Ana Celma, Irene Bijnsdorp, Alex Campos, Primiano Di Mauro, Salvador Borrós, Jaume Reventós 10 , Andreas Doll, Rosanna Paciucci, Michiel Pegtel 11 , Inés de Torres, Eduard Sabidó, Juan Morote, Mireia Olivan Journal Oncotarget Abstract Rapid and reliable diagnosis of prostate cancer (PCa) is highly desirable as current used methods la (show more...)Rapid and reliable diagnosis of prostate cancer (PCa) is highly desirable as current used methods lack specificity. In addition, identification of PCa biomarkers that can classify patients into high- and low-risk groups for disease progression at early stage will improve treatment decision-making. Here, we describe a set of protein-combination panels in urinary extracellular vesicles (EVs), defined by targeted proteomics and immunoblotting techniques that improve early non-invasive detection and stratification of PCa patients.We report a two-protein combination in urinary EVs that classifies benign and PCa patients (ADSV-TGM4), and a combination of five proteins able to significantly distinguish between high- and low-grade PCa patients (CD63-GLPK5-SPHM-PSA-PAPP). Proteins composing the panels were validated by immunohistochemistry assays in tissue microarrays (TMAs) confirming a strong link between the urinary EVs proteome and alterations in PCa tissues. Moreover, ADSV and TGM4 abundance yielded a high diagnostic potential in tissue and promising TGM4 prognostic power. These results suggest that the proteins identified in urinary EVs distinguishing high- and low grade PCa are a reflection of histological changes that may be a consequence of their functional involvement in PCa development. In conclusion, our study resulted in the identification of protein-combination panels present in urinary EVs that exhibit high sensitivity and specificity for PCa detection and patient stratification. Moreover, our study highlights the potential of targeted proteomic approaches-such as selected reaction monitoring (SRM)-as diagnostic assay for liquid biopsies via urinary EVs to improve diagnosis and prognosis of suspected PCa patients. (hide) EV-METRIC 22% (49th 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 High grade prostate 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 (d)(U)C Filtration Protein markers EV: SPHM/ TSG101/ CD63/ CD81/ ADSV non-EV: None Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens 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 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 60 Wash: Rotor Type Not specified Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ ADSV/ TSG101 Not detected EV-associated proteins SPHM Proteomics database Yes: ProteomeXchange Consortiu Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV210188
species	Homo sapiens
sample type	Urine
condition	Control condition	Low grade prostate cancer	High grade prostate cancer
separation protocol	(d)(U)C Filtration	(d)(U)C Filtration	(d)(U)C Filtration
Exp. nr.	2	1	3
EV-METRIC %	33	22	22