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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Isolation protocol	First author	Year	EV-METRIC
	EV120041	1/1	Bos bovis	Milk	0.2 µm filter Density cushion dUC Sucrose-DG	Reinhardt TA	2012	56%
Study summary Full title Bovine milk exosome proteome All authors Reinhardt TA, Lippolis JD, Nonnecke BJ, Sacco RE Journal J Proteomics Abstract Exosomes are 40-100 nm membrane vesicles of endocytic origin, secreted by cells and are found in bio (show more...)Exosomes are 40-100 nm membrane vesicles of endocytic origin, secreted by cells and are found in biological fluids including milk. These exosomes are extracellular organelles important in intracellular communication, and immune function. Therefore, the proteome of bovine milk exosomes may provide insight into the complex processes of milk production. Exosomes were isolated from the milk of mid-lactation cows. Purified exosomes were trypsin digested, subjected offline high pH reverse phase chromatography and further fractionated on a nanoLC connected to tandem mass spectrometer. This resulted in identification of 2107 proteins that included all of the major exosome protein markers. The major milk fat globule membrane (MFGM) proteins (Butyrophilin, Xanthine oxidase, Adipophilin and Lactadherin) were the most abundant proteins found in milk exosomes. However, they represented only 0.4-1.2% of the total spectra collected from milk exosomes compared to 15-28% of the total spectra collected in the MFGM proteome. These data show that the milk exosome secretion pathway differs significantly from that of the MFGM in part due to the greatly reduced presence of MFGM proteins. The protein composition of milk exosomes provides new information on milk protein composition and the potential physiological significance of exosomes to mammary physiology. (hide) EV-METRIC 56% (93rd 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 Milk Focus vesicles exosomes 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 0.2 µm filter + Density cushion + dUC + Sucrose-DG Adj. k-factor 157.1 (pelleting) Protein markers EV: TSG101/ MFGE8 non-EV: None Proteomics yes EV density (g/ml) 1.7 Show all info Study aim Omics Sample Species Bos bovis Sample Type Milk Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 60 Pelleting: rotor type 50.2Ti Pelleting: adjusted k-factor 157.1 Density gradient Density medium Sucrose Lowest density fraction 5 Highest density fraction 43 Orientation Bottom-up Rotor type 50.2Ti Speed (g) 200000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins TSG101/ MFGE8 ELISA Detected EV-associated proteins MFGE8 Fluorescent NTA Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

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