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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV120083	1/1	Homo sapiens	Urine	(d)(U)C DC	Raj DA	2012	33%
Study summary Full title A multiplex quantitative proteomics strategy for protein biomarker studies in urinary exosomes All authors Raj DA, Fiume I, Capasso G, Pocsfalvi G Journal Kidney Int Abstract Urinary exosomes have received considerable attention as a potential biomarker source for the diagno (show more...)Urinary exosomes have received considerable attention as a potential biomarker source for the diagnosis of renal diseases. Notwithstanding, their use in protein biomarker research is hampered by the lack of efficient methods for vesicle isolation, lysis, and protein quantification. Here we report an improved ultracentrifugation-based method that facilitates the solubilization and removal of major impurities associated with urinary exosomes. A double-cushion sucrose/D(2)O centrifugation step was used after a two-step differential centrifugation to separate exosomes from the heavier vesicles. After the removal of uromodulin, 378 and 79 unique proteins were identified, respectively, in low- and high-density fractions. Comparison of our data with two previously published data sets helped to define proteins commonly found in urinary exosomes. Lysis, protein extraction, and in-solution digestion of exosomes were then optimized for MudPIT application. More than a hundred exosomal proteins were quantified by four-plex iTRAQ analysis of single and pooled samples from two different age groups. For healthy men, six proteins (TSN1, PODXL, IDHC, PPAP, ACBP, and ANXA5) showed significant expression differences between exosome pools of those aged 25-50 and 50-70 years old. Thus, exosomes isolated by our method provide the basis for the development of robust quantitative methods for protein biomarker research. (hide) EV-METRIC 33% (65th 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 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 DC Adj. k-factor 78.45 (pelleting) Protein markers EV: Alix/ TSG101/ Beta-actin/ AQP2/ CD9 non-EV: uromodulin Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g 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) 60 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 78.45 Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD9/ TSG101/ AQP2/ Beta-actin ELISA Detected EV-associated proteins AQP2/ Beta-actin Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

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EV-TRACK ID	EV120083
species	Homo sapiens
sample type	Urine
condition	NAY
separation protocol	(d)(U)C DC
Exp. nr.	1
EV-METRIC %	33