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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140003	1/1	Homo sapiens	Urine	(d)(U)C DG	Hogan MC	2014	67%
Study summary Full title Subfractionation, characterization, and in-depth proteomic analysis of glomerular membrane vesicles in human urine All authors Hogan MC, Johnson KL, Zenka RM, Charlesworth MC, Madden BJ, Mahoney DW, Oberg AL, Huang BQ, Leontovich AA, Nesbitt LL, Bakeberg JL, McCormick DJ, Bergen HR, Ward CJ Journal Kidney Int Abstract Urinary exosome-like vesicles (ELVs) are a heterogenous mixture (diameter 40-200 nm) containing vesi (show more...)Urinary exosome-like vesicles (ELVs) are a heterogenous mixture (diameter 40-200 nm) containing vesicles shed from all segments of the nephron including glomerular podocytes. Contamination with Tamm-Horsfall protein (THP) oligomers has hampered their isolation and proteomic analysis. Here we improved ELV isolation protocols employing density centrifugation to remove THP and albumin, and isolated a glomerular membranous vesicle (GMV)-enriched subfraction from 7 individuals identifying 1830 proteins and in 3 patients with glomerular disease identifying 5657 unique proteins. The GMV fraction was composed of podocin/podocalyxin-positive irregularly shaped membranous vesicles and podocin/podocalyxin-negative classical exosomes. Ingenuity pathway analysis identified integrin, actin cytoskeleton, and Rho GDI signaling in the top three canonical represented signaling pathways and 19 other proteins associated with inherited glomerular diseases. The GMVs are of podocyte origin and the density gradient technique allowed isolation in a reproducible manner. We show many nephrotic syndrome proteins, proteases, and complement proteins involved in glomerular disease are in GMVs and some were only shed in the disease state (nephrin, TRPC6, INF2 and phospholipase A2 receptor). We calculated sample sizes required to identify new glomerular disease biomarkers, expand the ELV proteome, and provide a reference proteome in a database that may prove useful in the search for biomarkers of glomerular disease. (hide) EV-METRIC 67% (94th 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 Membrane(-derived) 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 DG Protein markers EV: Polycystin1/ Podocin non-EV: Tamm-Horsfall glycoprotein Proteomics yes EV density (g/ml) 1.055 TEM measurements 91.4(median) 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 Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Density gradient Lowest density fraction 5 Highest density fraction 30 Orientation Top-down Speed (g) 100000 Characterization: Protein analysis Western Blot Detected EV-associated proteins Podocin/ Polycystin1 Detected contaminants Tamm-Horsfall glycoprotein ELISA Detected EV-associated proteins Podocin/ Polycystin1 Characterization: Particle analysis EM EM-type transmission EM/ immune EM EM protein Podocalyxin; Podocin Image type Close-up, Wide-field Report size (nm) 91.4(median)

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EV-TRACK ID	EV140003
species	Homo sapiens
sample type	Urine
condition	NAY
separation protocol	(d)(U)C DG
Exp. nr.	1
EV-METRIC %	67