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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230635	1/2	Porphyromonas gingivalis	ATCC 33277	(d)(U)C Filtration	Bai D	2015	33%
Study summary Full title Immunoreactive antigens recognized in serum samples from mice intranasally immunized with Porphyromonas gingivalis outer membrane vesicles. All authors Bai D, Nakao R, Ito A, Uematsu H, Senpuku H Journal Pathog Dis Abstract Outer membrane vesicles (OMVs) of the periodontopathic bacterium Porphyromonas gingivalis contain a (show more...)Outer membrane vesicles (OMVs) of the periodontopathic bacterium Porphyromonas gingivalis contain a wide range of virulence factors including lipopolysaccharide (LPS), fimbriae and gingipains. We have recently reported strong immunogenicity of OMVs using an intranasal vaccine mouse model. In the present study, we performed sub-immunoproteome analysis of OMV-immunized mouse serum samples from six different mice in order to identify immunodominant antigens. The combination of two-dimensional (2D) gel electrophoresis and mass spectrometry analysis identified OMV proteins of 53 spots on a 2D map, and it was notable that OMV proteins were largely distributed within a low pH range, in marked contrast to the ubiquitous distribution of outer membrane proteins. Western blot using the six serum samples after 2D electrophoresis revealed that all showed immunoreactivity to some diffuse signals at extremely low pH, which was similar to the distribution of immunoreactive signals when the A-LPS antibody was used. Mass spectrometry analysis also demonstrated that the signals corresponded to a wide range of virulence factors including A-LPS-modified proteins such as gingipains. Absorption of serum with LPS resulted in a dramatic reduction of immmunoreactivity. We conclude that LPS and A-LPS-modified proteins in OMVs carry immunodominant determinants and eventually elicit P. gingivalis-specific antibodies in mice. (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 Filtration Protein markers EV: RgpA/ FimA/ Mfa1 non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches)/vaccine development Sample Species Porphyromonas gingivalis Sample Type Cell culture supernatant EV-producing cells ATCC 33277 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 180 Pelleting: rotor type Type 41 Ti Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins RgpA/ FimA/ Mfa1 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field Report size (nm) 50-80

	EV230635	2/2	Porphyromonas gingivalis	ATCC 33277	(d)(U)C Filtration	Bai D	2015	14%
Study summary Full title Immunoreactive antigens recognized in serum samples from mice intranasally immunized with Porphyromonas gingivalis outer membrane vesicles. All authors Bai D, Nakao R, Ito A, Uematsu H, Senpuku H Journal Pathog Dis Abstract Outer membrane vesicles (OMVs) of the periodontopathic bacterium Porphyromonas gingivalis contain a (show more...)Outer membrane vesicles (OMVs) of the periodontopathic bacterium Porphyromonas gingivalis contain a wide range of virulence factors including lipopolysaccharide (LPS), fimbriae and gingipains. We have recently reported strong immunogenicity of OMVs using an intranasal vaccine mouse model. In the present study, we performed sub-immunoproteome analysis of OMV-immunized mouse serum samples from six different mice in order to identify immunodominant antigens. The combination of two-dimensional (2D) gel electrophoresis and mass spectrometry analysis identified OMV proteins of 53 spots on a 2D map, and it was notable that OMV proteins were largely distributed within a low pH range, in marked contrast to the ubiquitous distribution of outer membrane proteins. Western blot using the six serum samples after 2D electrophoresis revealed that all showed immunoreactivity to some diffuse signals at extremely low pH, which was similar to the distribution of immunoreactive signals when the A-LPS antibody was used. Mass spectrometry analysis also demonstrated that the signals corresponded to a wide range of virulence factors including A-LPS-modified proteins such as gingipains. Absorption of serum with LPS resulted in a dramatic reduction of immmunoreactivity. We conclude that LPS and A-LPS-modified proteins in OMVs carry immunodominant determinants and eventually elicit P. gingivalis-specific antibodies in mice. (hide) EV-METRIC 14% (44th 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 mfa1 K.O. 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 Filtration Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches)/vaccine development Sample Species Porphyromonas gingivalis Sample Type Cell culture supernatant EV-producing cells ATCC 33277 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 180 Pelleting: rotor type Type 41 Ti Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis None Protein Concentration Method Bradford Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV230635
species	Porphyromonas gingivalis
sample type	Cell culture
cell type	ATCC 33277
condition	Control condition	mfa1 K.O.
separation protocol	dUC/ Filtration	dUC/ Filtration
Exp. nr.	1	2
EV-METRIC %	33	14