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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV180060	1/2	Homo sapiens	NA	(d)(U)C SEC UF	Benedikter BJ	2019	57%
Study summary Full title Proteomic analysis reveals procoagulant properties of cigarette smoke-induced extracellular vesicles. All authors Benedikter BJ, Bouwman FG, Heinzmann ACA, Vajen T, Mariman EC, Wouters EFM, Savelkoul PHM, Koenen RR, Rohde GGU, van Oerle R, Spronk HM, Stassen FRM Journal J Extracell Vesicles Abstract Airway epithelial cells secrete extracellular vesicles (EVs) under basal conditions and when exposed (show more...)Airway epithelial cells secrete extracellular vesicles (EVs) under basal conditions and when exposed to cigarette smoke extract (CSE). Getting insights into the composition of these EVs will help unravel their functions in homeostasis and smoking-induced pathology. Here, we characterized the proteomic composition of basal and CSE-induced airway epithelial EVs. BEAS-2B cells were left unexposed or exposed to 1% CSE for 24 h, followed by EV isolation using ultrafiltration and size exclusion chromatography. Isolated EVs were labelled with tandem mass tags and their proteomic composition was determined using nano-LC-MS/MS. Tissue factor (TF) activity was determined by a factor Xa generation assay, phosphatidylserine (PS) content by prothrombinase assay and thrombin generation using calibrated automated thrombogram (CAT). Nano-LC-MS/MS identified 585 EV-associated proteins with high confidence. Of these, 201 were differentially expressed in the CSE-EVs according to the moderated t-test, followed by false discovery rate (FDR) adjustment with the FDR threshold set to 0.1. Functional enrichment analysis revealed that 24 proteins of the pathway haemostasis were significantly up-regulated in CSE-EVs, including TF. Increased TF expression on CSE-EVs was confirmed by bead-based flow cytometry and was associated with increased TF activity. CSE-EVs caused faster and more thrombin generation in normal human plasma than control-EVs, which was partly TF-, but also PS-dependent. In conclusion, proteomic analysis allowed us to predict procoagulant properties of CSE-EVs which were confirmed in vitro. Cigarette smoke-induced EVs may contribute to the increased cardiovascular and respiratory risk observed in smokers. (hide) EV-METRIC 57% (88th 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 NA Sample origin Control condition Focus 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 SEC UF Protein markers EV: CD63/ MFGE8/ CD81/ TF/ HSP70/ CD9 non-EV: Proteomics yes Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type NA EV-producing cells BEAS-2B EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 80 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5 Resin type Sepharose CL-4B Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Detected EV-associated proteins HSP70/ CD81/ MFGE8/ CD63 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins TF/ CD63/ CD81/ CD9 Proteomics database Yes Characterization: Lipid analysis Yes Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 80-250 EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 70

	EV180060	2/2	Homo sapiens	NA	(d)(U)C SEC UF	Benedikter BJ	2019	57%
Study summary Full title Proteomic analysis reveals procoagulant properties of cigarette smoke-induced extracellular vesicles. All authors Benedikter BJ, Bouwman FG, Heinzmann ACA, Vajen T, Mariman EC, Wouters EFM, Savelkoul PHM, Koenen RR, Rohde GGU, van Oerle R, Spronk HM, Stassen FRM Journal J Extracell Vesicles Abstract Airway epithelial cells secrete extracellular vesicles (EVs) under basal conditions and when exposed (show more...)Airway epithelial cells secrete extracellular vesicles (EVs) under basal conditions and when exposed to cigarette smoke extract (CSE). Getting insights into the composition of these EVs will help unravel their functions in homeostasis and smoking-induced pathology. Here, we characterized the proteomic composition of basal and CSE-induced airway epithelial EVs. BEAS-2B cells were left unexposed or exposed to 1% CSE for 24 h, followed by EV isolation using ultrafiltration and size exclusion chromatography. Isolated EVs were labelled with tandem mass tags and their proteomic composition was determined using nano-LC-MS/MS. Tissue factor (TF) activity was determined by a factor Xa generation assay, phosphatidylserine (PS) content by prothrombinase assay and thrombin generation using calibrated automated thrombogram (CAT). Nano-LC-MS/MS identified 585 EV-associated proteins with high confidence. Of these, 201 were differentially expressed in the CSE-EVs according to the moderated t-test, followed by false discovery rate (FDR) adjustment with the FDR threshold set to 0.1. Functional enrichment analysis revealed that 24 proteins of the pathway haemostasis were significantly up-regulated in CSE-EVs, including TF. Increased TF expression on CSE-EVs was confirmed by bead-based flow cytometry and was associated with increased TF activity. CSE-EVs caused faster and more thrombin generation in normal human plasma than control-EVs, which was partly TF-, but also PS-dependent. In conclusion, proteomic analysis allowed us to predict procoagulant properties of CSE-EVs which were confirmed in vitro. Cigarette smoke-induced EVs may contribute to the increased cardiovascular and respiratory risk observed in smokers. (hide) EV-METRIC 57% (88th 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 NA Sample origin 1% cigarette smoke extract Focus 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 SEC UF Protein markers EV: TF/ CD81/ CD63/ CD9 non-EV: Proteomics yes Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type NA EV-producing cells BEAS-2B EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 80 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5 Resin type Sepharose CL-4B Characterization: Protein analysis Protein Concentration Method Bradford Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins TF/ CD63/ CD81/ CD9 Proteomics database Yes Characterization: Lipid analysis Yes Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 80-250 EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 65

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EV-TRACK ID	EV180060
species	Homo sapiens
cell type	BEAS-2B
condition	Control condition	1% cigarette smoke extract
separation protocol	(d)(U)C SEC UF	(d)(U)C SEC UF
Exp. nr.	1	2
EV-METRIC %	57	57