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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV120003	1/2	Trypanosoma cruzi	Protozoa	(d)(U)C DG Filtration	Bayer-Santos E	2012	78%
Study summary Full title Proteomic analysis of Trypanosoma cruzi secretome: characterization of two populations of extracellular vesicles and soluble proteins All authors Bayer-Santos E, Aguilar-Bonavides C, Rodrigues SP, Cordero EM, Marques AF, Varela-Ramirez A, Choi H, Yoshida N, da Silveira JF, Almeida IC Journal J Proteome Res Abstract Microorganisms use specialized systems to export virulence factors into host cells. Secretion of eff (show more...)Microorganisms use specialized systems to export virulence factors into host cells. Secretion of effector proteins into the extracellular environment has been described in Trypanosoma cruzi; however, a comprehensive proteomic analysis of the secretome and the secretion mechanisms involved remain elusive. Here, we present evidence that T. cruzi releases proteins associated with vesicles that are formed by at least two different mechanisms. Transmission electron microscopy showed larger vesicles budding from the plasma membrane of noninfective epimastigotes and infective metacyclic trypomastigotes, as well as smaller vesicles within the flagellar pocket of both forms. Parasite conditioned culture supernatant was fractionated and characterized by morphological, immunochemical, and proteomic analyses. Three fractions were obtained by differential ultracentrifugation: the first enriched in larger vesicles resembling ectosomes, the second enriched in smaller vesicles resembling exosomes, and a third fraction enriched in soluble proteins not associated with extracellular vesicles. Label-free quantitative proteomic analysis revealed a rich collection of proteins involved in metabolism, signaling, nucleic acid binding, and parasite survival and virulence. These findings support the notion that T. cruzi uses different secretion pathways to excrete/secrete proteins. Moreover, our results suggest that metacyclic forms may use extracellular vesicles to deliver cargo into host cells. (hide) EV-METRIC 78% (85th 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 Protozoa Sample origin NAY Focus vesicles extracellular 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 DG Filtration Adj. k-factor 240.8 (pelleting) Protein markers EV: FCaBP/ GP82/ GP35/50 non-EV: Proteomics yes EV density (g/ml) 1.08-1.14 Show all info Study aim Omics Sample Species Trypanosoma cruzi Sample Type Protozoa 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) 120 Pelleting: rotor type TH641 Pelleting: adjusted k-factor 240.8 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Bottom-up Speed (g) 200000 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins GP82/ GP35/50/ FCaBP ELISA Antibody details provided? No Detected EV-associated proteins GP82/ GP35/50/ FCaBP Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up, Wide-field

	EV120003	2/2	Trypanosoma cruzi	Protozoa	(d)(U)C DG Filtration	Bayer-Santos E	2012	78%
Study summary Full title Proteomic analysis of Trypanosoma cruzi secretome: characterization of two populations of extracellular vesicles and soluble proteins All authors Bayer-Santos E, Aguilar-Bonavides C, Rodrigues SP, Cordero EM, Marques AF, Varela-Ramirez A, Choi H, Yoshida N, da Silveira JF, Almeida IC Journal J Proteome Res Abstract Microorganisms use specialized systems to export virulence factors into host cells. Secretion of eff (show more...)Microorganisms use specialized systems to export virulence factors into host cells. Secretion of effector proteins into the extracellular environment has been described in Trypanosoma cruzi; however, a comprehensive proteomic analysis of the secretome and the secretion mechanisms involved remain elusive. Here, we present evidence that T. cruzi releases proteins associated with vesicles that are formed by at least two different mechanisms. Transmission electron microscopy showed larger vesicles budding from the plasma membrane of noninfective epimastigotes and infective metacyclic trypomastigotes, as well as smaller vesicles within the flagellar pocket of both forms. Parasite conditioned culture supernatant was fractionated and characterized by morphological, immunochemical, and proteomic analyses. Three fractions were obtained by differential ultracentrifugation: the first enriched in larger vesicles resembling ectosomes, the second enriched in smaller vesicles resembling exosomes, and a third fraction enriched in soluble proteins not associated with extracellular vesicles. Label-free quantitative proteomic analysis revealed a rich collection of proteins involved in metabolism, signaling, nucleic acid binding, and parasite survival and virulence. These findings support the notion that T. cruzi uses different secretion pathways to excrete/secrete proteins. Moreover, our results suggest that metacyclic forms may use extracellular vesicles to deliver cargo into host cells. (hide) EV-METRIC 78% (85th 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 Protozoa Sample origin NAY Focus vesicles extracellular 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 DG Filtration Adj. k-factor 240.8 (pelleting) Protein markers EV: FCaBP/ GP82/ GP35/50 non-EV: Proteomics yes EV density (g/ml) 1.14-1.2 Show all info Study aim Omics Sample Species Trypanosoma cruzi Sample Type Protozoa 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) 960 Pelleting: rotor type TH641 Pelleting: adjusted k-factor 240.8 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Bottom-up Speed (g) 200000 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins GP82/ GP35/50/ FCaBP ELISA Antibody details provided? No Detected EV-associated proteins GP82/ GP35/50/ FCaBP Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up, Wide-field

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EV-TRACK ID	EV120003
species	Trypanosoma cruzi
sample type	Protozoa
condition	NAY
separation protocol	(d)(U)C DG Filtration	(d)(U)C DG Filtration
Exp. nr.	1	2
EV-METRIC %	78	78