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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210326	1/1	Spodoptera frugiperda	Midgut epithelium	(d)(U)C DG	Fuzita FJ	2019	57%
Study summary Full title Detergent-resistant domains in Spodoptera frugiperda midgut microvillar membranes and their relation to microapocrine secretion. All authors Fuzita FJ, Pimenta DC, Palmisano G, Terra WR, Ferreira C Journal Comp Biochem Physiol B Biochem Mol Biol Abstract The midgut from lepidopteran insects has a particular way to release proteins to the lumen, named mi (show more...)The midgut from lepidopteran insects has a particular way to release proteins to the lumen, named microapocrine secretion that could be an adaptation to release secretory contents into the lumen at water absorbing regions. In this process small vesicles (microapocrine vesicles) bud from the midgut microvilli as double membrane vesicles, where the inner membrane comes from the secretion vesicle and the outer one from the microvillar membrane. The molecular machinery associated with this process may be recruited by specific midgut microvilli membrane domains. To address to this, Spodoptera frugiperda midgut microvillar membranes, prepared by magnesium treatment and free from cytoskeleton with the hyperosmotic Tris procedure, were submitted to detergent extraction and fractionated by density gradient ultracentrifugation. Detergent-resistant membrane domains (DRM) were recovered and their proteins identified by proteomics. Microapocrine vesicles were isolated by washing the luminal surface of the midgut epithelium, followed by freezing and thawing plus centrifugation to recover only membranes. Proteins from purified microvillar membranes and microapocrine vesicle membranes were identified by proteomics. Comparison of the two populations suggests that the budding of microapocrine vesicles surrounded by microvillar membrane is not a random process, because only around 50% of the microvillar membrane proteins are in the microapocrine vesicles. From the 16 proteins from DRM, 14 were enriched in the microapocrine membrane vesicles. These results suggest that on budding, the microapocrine vesicle membrane is enclosed by DRM and a surrounding area of the microvillar membrane. It is proposed that the DRMs somehow recruit the proteins composing the secretory machinery. (hide) EV-METRIC 57% (50th 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 Midgut epithelium Sample origin Control condition Focus vesicles microapocrine 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 (Differential) (ultra)centrifugation Density gradient Protein markers EV: None non-EV: None Proteomics yes EV density (g/ml) 1.13-1.24 Show all info Study aim Function/ Identification of content (omics approaches) Sample Species Spodoptera frugiperda Sample Type Midgut epithelium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type P40ST Pelleting: speed (g) 100000 Density gradient Type Continuous Lowest density fraction 25 Highest density fraction 60 Total gradient volume, incl. sample (mL) Not reported Sample volume (mL) 2 Orientation Top-down Rotor type P40ST Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing None Characterization: Protein analysis Protein Concentration Method BCA Proteomics database No Characterization: Lipid analysis No

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EV-TRACK ID	EV210326
species	Spodoptera frugiperda
sample type	Midgut epithelium
condition	Control condition
separation protocol	dUC/ Density gradient
Exp. nr.	1
EV-METRIC %	57