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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220200	2/3	Homo sapiens	Urine	(d)(U)C	Chutipongtanate S	2018	44%
Study summary Full title Multiplex Biomarker Screening Assay for Urinary Extracellular Vesicles Study: A Targeted Label-Free Proteomic Approach. All authors Chutipongtanate S, Greis KD Journal Sci Rep Abstract The recent advance in targeted label-free proteomics, SWATH-MS, can provide consistent protein detec (show more...)The recent advance in targeted label-free proteomics, SWATH-MS, can provide consistent protein detection and reproducible protein quantitation, which is a considerable advantage for biomarker study of urinary extracellular vesicles. We developed a SWATH-MS workflow with a curated spectral library of 1,145 targets. Application of the workflow across nine replicates of three sample types (exosome-like vesicles (ELVs), microvesicles (MVs) and urine proteins (UPs)) resulted in the quantitation of 888 proteins at FDR <1%. The median-coefficient of variation of the 888 proteins in the ELV sample was 7.7%, indicating excellent reproducibility. Data analysis showed common exosome markers, (i.e. CD9, CD63, ALIX, TSG101 and HSP70) were enriched in urinary ELVs as compared to MVs and UPs. The use of a multiplex biomarker screening assay focused on ELVs was investigated, and perspectives in future applications are discussed. (hide) EV-METRIC 44% (80th 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 (shedding) microvesicle 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: None non-EV: None Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type SW 60 Ti Pelleting: speed (g) 100000 EV-subtype Distinction between multiple subtypes Centrifugation speed/ Other Used subtypes Microvesicles Characterization: Protein analysis Protein Concentration Method Pierce660 assay Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins HSP70 Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes EM EM-type Transmission-EM Image type Close-up Report size (nm) > 100 nm

	EV220200	3/3	Homo sapiens	Urine	(d)(U)C	Chutipongtanate S	2018	44%
Study summary Full title Multiplex Biomarker Screening Assay for Urinary Extracellular Vesicles Study: A Targeted Label-Free Proteomic Approach. All authors Chutipongtanate S, Greis KD Journal Sci Rep Abstract The recent advance in targeted label-free proteomics, SWATH-MS, can provide consistent protein detec (show more...)The recent advance in targeted label-free proteomics, SWATH-MS, can provide consistent protein detection and reproducible protein quantitation, which is a considerable advantage for biomarker study of urinary extracellular vesicles. We developed a SWATH-MS workflow with a curated spectral library of 1,145 targets. Application of the workflow across nine replicates of three sample types (exosome-like vesicles (ELVs), microvesicles (MVs) and urine proteins (UPs)) resulted in the quantitation of 888 proteins at FDR <1%. The median-coefficient of variation of the 888 proteins in the ELV sample was 7.7%, indicating excellent reproducibility. Data analysis showed common exosome markers, (i.e. CD9, CD63, ALIX, TSG101 and HSP70) were enriched in urinary ELVs as compared to MVs and UPs. The use of a multiplex biomarker screening assay focused on ELVs was investigated, and perspectives in future applications are discussed. (hide) EV-METRIC 44% (80th 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 Other/ Exosome-like 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: None non-EV: None Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type SW 60 Ti Pelleting: speed (g) 100000 EV-subtype Distinction between multiple subtypes Centrifugation speed Used subtypes Exosome-like Vesicles (Centrifugation speed Characterization: Protein analysis Protein Concentration Method Pierce660 assay Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Alix/ TSG101/ HSP70 Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes EM EM-type Transmission-EM Image type Close-up Report size (nm) < 100 nm

	EV220200	1/3	Homo sapiens	MOLM-13	(d)(U)C	Chutipongtanate S	2018	33%
Study summary Full title Multiplex Biomarker Screening Assay for Urinary Extracellular Vesicles Study: A Targeted Label-Free Proteomic Approach. All authors Chutipongtanate S, Greis KD Journal Sci Rep Abstract The recent advance in targeted label-free proteomics, SWATH-MS, can provide consistent protein detec (show more...)The recent advance in targeted label-free proteomics, SWATH-MS, can provide consistent protein detection and reproducible protein quantitation, which is a considerable advantage for biomarker study of urinary extracellular vesicles. We developed a SWATH-MS workflow with a curated spectral library of 1,145 targets. Application of the workflow across nine replicates of three sample types (exosome-like vesicles (ELVs), microvesicles (MVs) and urine proteins (UPs)) resulted in the quantitation of 888 proteins at FDR <1%. The median-coefficient of variation of the 888 proteins in the ELV sample was 7.7%, indicating excellent reproducibility. Data analysis showed common exosome markers, (i.e. CD9, CD63, ALIX, TSG101 and HSP70) were enriched in urinary ELVs as compared to MVs and UPs. The use of a multiplex biomarker screening assay focused on ELVs was investigated, and perspectives in future applications are discussed. (hide) EV-METRIC 33% (75th 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 Other/ Exosome-like 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: None non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MOLM-13 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type SW 60 Ti Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Pierce660 assay Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins TSG101/ HSP70/ Alix Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Close-up Report size (nm) < 100 nm

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EV-TRACK ID	EV220200
species	Homo sapiens
sample type	Urine	Urine	Cell culture
cell type	NA	NA	MOLM-13
condition	Control condition	Control condition	Control condition
separation protocol	dUC	dUC	dUC
EV subtype	Microvesicles	Exosome-like Vesicles (Centrifugation speed	NA
vesicle related term	(shedding) microvesicle	Other/ Exosome-like Vesicle	Other/ Exosome-like Vesicle
Exp. nr.	2	3	1
EV-METRIC %	44	44	33