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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV120006	1/1	Homo sapiens	Urine	(d)(U)C	Musante L	2012	56%
Study summary Full title Biochemical and physical characterisation of urinary nanovesicles following CHAPS treatment All authors Musante L, Saraswat M, Duriez E, Byrne B, Ravidà A, Domon B, Holthofer H Journal PLoS One Abstract Urinary exosomes represent a precious source of potential biomarkers for disease biology. Currently, (show more...)Urinary exosomes represent a precious source of potential biomarkers for disease biology. Currently, the methods for vesicle isolation are severely restricted by the tendency of vesicle entrapment, e.g. by the abundant Tamm-Horsfall protein (THP) polymers. Treatment by reducing agents such as dithiothreitol (DTT) releases entrapped vesicles, thus increasing the final yield. However, this harsh treatment can cause remodelling of all those proteins which feature extra-vesicular domains stabilized by internal disulfide bridges and have detrimental effects on their biological activity. In order to optimize exosomal yield, we explore two vesicle treatment protocols - dithiothreitol (DTT) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic (CHAPS) - applied to the differential centrifugation protocol for exosomal vesicle isolation. The results show that CHAPS treatment does not affect vesicle morphology or exosomal marker distribution, thus eliminating most of THP interference. Moreover, the recovery and preservation of catalytic activity of two trans-membrane proteases, dipeptidyl peptidase IV and nephrilysin, was examined and found to be clearly superior after CHAPS treatment compared to DTT. Finally, proteomic profiling by mass spectrometry (MS) revealed that 76.2% of proteins recovered by CHAPS are common to those seen for DTT treatment, which illustrates underlining similarities between the two approaches. In conclusion, we provide a major improvement to currently-utilized urinary vesicle isolation strategies to allow recovery of urinary vesicles without the deleterious interference of abundant urinary proteins, while preserving typical protein folding and, consequently, the precious biological activity of urinary proteins which serve as valuable biomarkers. (hide) EV-METRIC 56% (90th 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 Urine Sample origin NAY Focus vesicles Nano(-sized) 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 Adj. k-factor 78.45 (pelleting) / 78.45 (washing) Protein markers EV: TSG101/ CD63/ MFGE8 non-EV: Tamm-Horsfall glycoproteinNephrin Proteomics yes Show all info Study aim Technical Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 78.45 Wash: volume per pellet (ml) 5 Wash: Rotor Type 70Ti Wash: adjusted k-factor 78.45 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ TSG101/ MFGE8 Detected contaminants Tamm-Horsfall glycoproteinNephrin ELISA Detected EV-associated proteins MFGE8 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

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EV-TRACK ID	EV120006
species	Homo sapiens
sample type	Urine
condition	NAY
separation protocol	(d)(U)C
Exp. nr.	1
EV-METRIC %	56