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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV130016	1/1	Homo sapiens	Urine	(d)(U)C DG	Raimondo F	2013	44%
Study summary Full title Differential protein profiling of renal cell carcinoma urinary exosomes All authors Raimondo F, Morosi L, Corbetta S, Chinello C, Brambilla P, Della Mina P, Villa A, Albo G, Battaglia C, Bosari S, Magni F, Pitto M Journal Mol Biosyst Abstract Renal cell carcinoma (RCC) accounts for about 3% of all human malignancies and its incidence is incr (show more...)Renal cell carcinoma (RCC) accounts for about 3% of all human malignancies and its incidence is increasing. There are no standard biomarkers currently used in the clinical management of patients with renal cell carcinoma. A promising strategy for new biomarker detection is comparative proteomics of urinary exosomes (UE), nanovesicles released by every epithelial cell facing the urinary space, enriched in renal proteins and excluding high-abundance plasmatic proteins, such as albumin. Aim of the work is to establish the protein profile of exosomes isolated from urines of RCC patient compared with control subjects. We enrolled 29 clear cell RCC patients and 23 control healthy subjects (CTRL), age and sex-matched, for urine collection and vesicle isolation by differential centrifugation. Such vesicles were morphologically and biochemically characterized and proved to share exosome properties. Proteomic analysis, performed on 9 urinary exosome (UE) pooled samples by gel based digestion followed by LC-MS/MS, led to the identification of 261 proteins from CTRL subject UE and 186 from RCC patient UE, and demonstrated that most of the identified proteins are membrane associated or cytoplasmic. Moreover, about a half of identified proteins are not shared between RCC and control UE. Starting from these observations, and from the literature, we selected a panel of 10 proteins, whose UE differential content was subjected to immunoblotting validation. Results show for the first time that RCC UE protein content is substantially and reproducibly different from control UE, and that these differences may provide clues for new RCC biomarker discovery. (hide) EV-METRIC 44% (81st 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 DG Protein markers EV: TSG101/ Flotilin1/ Syntenin/ AQP1/ DKK4/ EMMPRIN/ Podocalyxin/ CAIX/ CD9/ CD10/ MMP9/ CP/ DPEP1 non-EV: Proteomics yes EV density (g/ml) 1.100 TEM measurements 41.1+-12.7 Show all info Study aim Omics 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) 60 Density gradient Only used for validation of main results Yes Lowest density fraction 5 Highest density fraction 40 Orientation Top-down Speed (g) 100000 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD9/ Flotilin1/ Syntenin/ TSG101/ CD10/ EMMPRIN/ DPEP1/ CP/ MMP9/ Podocalyxin/ CAIX/ DKK4/ AQP1 ELISA Detected EV-associated proteins CD10/ EMMPRIN/ DPEP1/ CP/ MMP9/ Podocalyxin/ CAIX/ DKK4/ AQP1 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field Report size (nm) 41.1+-12.7

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EV-TRACK ID	EV130016
species	Homo sapiens
sample type	Urine
condition	NAY
separation protocol	(d)(U)C DG
Exp. nr.	1
EV-METRIC %	44