Search > Results

You searched for: EV120003 (EV-TRACK ID)

Showing 1 - 2 of 2

Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, isolation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Isolation protocol
  • Gives a short, non-chronological overview of the different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Experiment number
  • Experiments differ in Vesicle type
Experiment number
  • Experiments differ in Vesicle type
Details EV-TRACK ID Experiment nr. Species Sample type Isolation protocol First author Year EV-METRIC
EV120003 1/2 Trypanosoma cruzi Other 0.45 µm filter
dUC
Sucrose-DG (valid.)
Bayer-Santos E 2012 78%

Study summary

Full title
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% (99th 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Other
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.45 µm filter + dUC + Sucrose-DG (valid.)
Adj. k-factor
240.8 (pelleting)
Protein markers
EV: GP82/ GP35/50/ FCaBP
non-EV: None
Proteomics
yes
EV density (g/ml)
1.08-1.14
Show all info
Study aim
Omics
Sample
Species
Trypanosoma cruzi
Sample Type
Other
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
TH641
Pelleting: adjusted k-factor
240.8
Density gradient
Only used for validation of main results
1
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
Detected EV-associated proteins
GP82/ GP35/50/ FCaBP
ELISA
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 Other 0.45 µm filter
dUC
Sucrose-DG (valid.)
Bayer-Santos E 2012 78%

Study summary

Full title
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% (99th 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Other
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.45 µm filter + dUC + Sucrose-DG (valid.)
Adj. k-factor
240.8 (pelleting)
Protein markers
EV: GP82/ GP35/50/ FCaBP
non-EV: None
Proteomics
yes
EV density (g/ml)
1.14-1.2
Show all info
Study aim
Omics
Sample
Species
Trypanosoma cruzi
Sample Type
Other
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
960
Pelleting: rotor type
TH641
Pelleting: adjusted k-factor
240.8
Density gradient
Only used for validation of main results
1
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
Detected EV-associated proteins
GP82/ GP35/50/ FCaBP
ELISA
Detected EV-associated proteins
GP82/ GP35/50/ FCaBP
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
1 - 2 of 2