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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV130111	1/1	Bos bovis	Milk	(d)(U)C DG Filtration	Reinhardt TA	2013	43%
Study summary Full title Bovine milk proteome: quantitative changes in normal milk exosomes, milk fat globule membranes and whey proteomes resulting from Staphylococcus aureus mastitis All authors Reinhardt TA, Sacco RE, Nonnecke BJ, Lippolis JD Journal J Proteomics Abstract Milk protein expression in healthy cows and cows with mastitis will provide information important fo (show more...)Milk protein expression in healthy cows and cows with mastitis will provide information important for the dairy food industry and immune function in the mammary gland. To facilitate protein discovery, milk was fractioned into whey, milk fat globule membranes (MFGM) and exosomes from healthy and Staphylococcus aureus infected cows. Amine-reactive isobaric tags (iTRAQ) were used to quantify protein changes between milk fractions isolated from healthy and S. aureus infected cows. 2971 milk proteins were identified with a false discovery rate of 0.1%. Greater than 300 milk proteins associated with host defense were identified and 94 were significantly differentially regulated in S. aureus infected milk compared to their uninfected controls. These differentially regulated host defense proteins were selectively segregated in the 3 milk compartments examined. An example of this segregation of host defense proteins was the partitioning and high concentration of proteins indicative of neutrophil extracellular traps (NETs) formation in the MFGM preparations from S. aureus infected milk as compared to exosomes or whey. Protein composition changes found in milk exosomes, MFGM and whey during an infection provides new and comprehensive information on milk protein composition in general as well as changes occurring during an infection.BIOLOGICAL SIGNIFICANCE: The significance of this study is the identification and quantification of the individual components of the neutrophil extracellular traps (NET) functional proteome in an apparent stable complex with MFGM and/or milk fat globules during an intra-mammary infection. NETs could be functionally relevant in intra-mammary infection, as it is known that during an infection neutrophils ingest large amounts of milk fat that down regulates many of their traditional immune functions. Thus the presence of NETs in milk fat provides new insights to mammary immune function and suggests a role for NETs in clinical mastitis. These in vivo NETs can now be tested to determine if they retain functional antimicrobial activity when primarily associated with milk fat. Then we can estimate their real world functional relevance during an intra-mammary infection, which is one key to understanding clinical mastitis in dairy cows. (hide) EV-METRIC 43% (60th 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 Milk Sample origin NAY Focus vesicles exosomes 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 104.8 (pelleting) Protein markers EV: non-EV: Proteomics yes Show all info Study aim Biomarker Sample Species Bos bovis Sample Type Milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type 50.2TI Pelleting: adjusted k-factor 104.8 Density gradient Lowest density fraction 5 Highest density fraction 43 Orientation Bottom-up Speed (g) 200000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis

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EV-TRACK ID	EV130111
species	Bos bovis
sample type	Milk
condition	NAY
separation protocol	(d)(U)C DG Filtration
Exp. nr.	1
EV-METRIC %	43