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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230666	1/5	Aggregatibacter actinomycetemcomitans	Strain 173	(d)(U)C DG Filtration	Kieselbach T	2015	33%
Study summary Full title Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles. All authors Kieselbach T, Zijnge V, Granström E, Oscarsson J Journal PLoS One Abstract Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive fo (show more...)Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease. (hide) EV-METRIC 33% (74th 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 Outer membrane 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 Density gradient Filtration Adj. k-factor 1 (pelleting) / 1 (washing) Protein markers EV: GroEL non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Aggregatibacter actinomycetemcomitans Sample Type Cell culture supernatant EV-producing cells Strain 173 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 85000 Pelleting: adjusted k-factor 1,52E Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 85000 Wash: adjusted k-factor 1,52E Density gradient Type Discontinuous Number of initial discontinuous layers 7 Lowest density fraction 10% Highest density fraction 45% Total gradient volume, incl. sample (mL) 4.170 Sample volume (mL) 0.150 Orientation Bottom-up Speed (g) 180000 Duration (min) 180 Fraction volume (mL) 0.2 Fraction processing Centrifugation Pelleting: speed (g) 16000 Pelleting: adjusted k-factor 9,55E Pelleting-wash: speed (g) JA-18.1 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Spectrophotometry Western Blot Detected EV-associated proteins GroEL Proteomics database ProteomeXchange Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Atomic force microscopy Image type Wide-field

	EV230666	3/5	Aggregatibacter actinomycetemcomitans	D7SS	(d)(U)C DG Filtration	Kieselbach T	2015	29%
Study summary Full title Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles. All authors Kieselbach T, Zijnge V, Granström E, Oscarsson J Journal PLoS One Abstract Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive fo (show more...)Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease. (hide) EV-METRIC 29% (67th 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 delltxA delcdtABC Focus vesicles Outer membrane 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 Density gradient Filtration Adj. k-factor 1 (pelleting) / 1 (washing) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Aggregatibacter actinomycetemcomitans Sample Type Cell culture supernatant EV-producing cells D7SS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 85000 Pelleting: adjusted k-factor 1,52E Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 85000 Wash: adjusted k-factor 1,52E Density gradient Type Discontinuous Number of initial discontinuous layers 7 Lowest density fraction 10% Highest density fraction 45% Total gradient volume, incl. sample (mL) 4.170 Sample volume (mL) 0.150 Orientation Bottom-up Speed (g) 180000 Duration (min) 180 Fraction volume (mL) 0.2 Fraction processing Centrifugation Pelleting: speed (g) 16000 Pelleting: adjusted k-factor 9,55E Pelleting-wash: speed (g) JA-18.1 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis None Protein Concentration Method Spectrophotometry Characterization: Lipid analysis No Characterization: Particle analysis None

	EV230666	4/5	Aggregatibacter actinomycetemcomitans	D7SS	(d)(U)C DG Filtration	Kieselbach T	2015	29%
Study summary Full title Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles. All authors Kieselbach T, Zijnge V, Granström E, Oscarsson J Journal PLoS One Abstract Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive fo (show more...)Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease. (hide) EV-METRIC 29% (67th 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 delpal Focus vesicles Outer membrane 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 Density gradient Filtration Adj. k-factor 1 (pelleting) / 1 (washing) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Aggregatibacter actinomycetemcomitans Sample Type Cell culture supernatant EV-producing cells D7SS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 85000 Pelleting: adjusted k-factor 1,52E Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 85000 Wash: adjusted k-factor 1,52E Density gradient Type Discontinuous Number of initial discontinuous layers 7 Lowest density fraction 10% Highest density fraction 45% Total gradient volume, incl. sample (mL) 4.170 Sample volume (mL) 0.150 Orientation Bottom-up Speed (g) 180000 Duration (min) 180 Fraction volume (mL) 0.2 Fraction processing Centrifugation Pelleting: speed (g) 16000 Pelleting: adjusted k-factor 9,55E Pelleting-wash: speed (g) JA-18.1 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis None Protein Concentration Method Spectrophotometry Characterization: Lipid analysis No Characterization: Particle analysis None

	EV230666	5/5	Aggregatibacter actinomycetemcomitans	JP2	(d)(U)C DG Filtration	Kieselbach T	2015	29%
Study summary Full title Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles. All authors Kieselbach T, Zijnge V, Granström E, Oscarsson J Journal PLoS One Abstract Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive fo (show more...)Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease. (hide) EV-METRIC 29% (67th 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 Outer membrane 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 Density gradient Filtration Adj. k-factor 1 (pelleting) / 1 (washing) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Aggregatibacter actinomycetemcomitans Sample Type Cell culture supernatant EV-producing cells JP2 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 85000 Pelleting: adjusted k-factor 1,52E Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 85000 Wash: adjusted k-factor 1,52E Density gradient Type Discontinuous Number of initial discontinuous layers 7 Lowest density fraction 10% Highest density fraction 45% Total gradient volume, incl. sample (mL) 4.170 Sample volume (mL) 0.150 Orientation Bottom-up Speed (g) 180000 Duration (min) 180 Fraction volume (mL) 0.2 Fraction processing Centrifugation Pelleting: speed (g) 16000 Pelleting: adjusted k-factor 9,55E Pelleting-wash: speed (g) JA-18.1 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis None Protein Concentration Method Spectrophotometry Characterization: Lipid analysis No Characterization: Particle analysis None

	EV230666	2/5	Aggregatibacter actinomycetemcomitans	D7SS	(d)(U)C DG Filtration	Kieselbach T	2015	22%
Study summary Full title Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles. All authors Kieselbach T, Zijnge V, Granström E, Oscarsson J Journal PLoS One Abstract Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive fo (show more...)Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease. (hide) EV-METRIC 22% (58th 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 Outer membrane 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 Density gradient Filtration Adj. k-factor 1 (pelleting) / 1 (washing) Protein markers EV: GroEL non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Aggregatibacter actinomycetemcomitans Sample Type Cell culture supernatant EV-producing cells D7SS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 85000 Pelleting: adjusted k-factor 1,52E Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 85000 Wash: adjusted k-factor 1,52E Density gradient Type Discontinuous Number of initial discontinuous layers 7 Lowest density fraction 10% Highest density fraction 45% Total gradient volume, incl. sample (mL) 4.170 Sample volume (mL) 0.150 Orientation Bottom-up Speed (g) 180000 Duration (min) 180 Fraction volume (mL) 0.2 Fraction processing Centrifugation Pelleting: speed (g) 16000 Pelleting: adjusted k-factor 9,55E Pelleting-wash: speed (g) JA-18.1 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Spectrophotometry Western Blot Detected EV-associated proteins GroEL Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV230666
species	Aggregatibacter actinomycetemcomitans
sample type	Cell culture
cell type	Strain 173	D7SS	D7SS	JP2	D7SS
condition	Control condition	delltxA delcdtABC	delpal	Control condition	Control condition
separation protocol	dUC/ Density gradient/ Filtration	dUC/ Density gradient/ Filtration	dUC/ Density gradient/ Filtration	dUC/ Density gradient/ Filtration	dUC/ Density gradient/ Filtration
Exp. nr.	1	3	4	5	2
EV-METRIC %	33	29	29	29	22