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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210199	1/1	Homo sapiens	Urine	(d)(U)C Filtration	Correll, Vanessa	2022	78%
Study summary Full title Optimization of small extracellular vesicle isolation from expressed prostatic secretions in urine for in-depth proteomic analysis All authors Vanessa L. Correll, Joseph J. Otto, Cristina M. Risi, Brian P. Main, Paul C. Boutros, Thomas Kislinger, Vitold E. Galkin, Julius O. Nyalwidhe, O. John Semmes, Lifang Yang Journal J Extracell Vesicles Abstract The isolation and subsequent molecular analysis of extracellular vesicles (EVs) derived from patient (show more...)The isolation and subsequent molecular analysis of extracellular vesicles (EVs) derived from patient samples is a widely used strategy to understand vesicle biology and to facilitate biomarker discovery. Expressed prostatic secretions in urine are a tumor proximal fluid that has received significant attention as a source of potential prostate cancer (PCa) biomarkers for use in liquid biopsy protocols. Standard EV isolation methods like differential ultracentrifugation (dUC) co-isolate protein contaminants that mask lower-abundance proteins in typical mass spectrometry (MS) protocols. Further complicating the analysis of expressed prostatic secretions, uromodulin, also known as Tamm-Horsfall protein (THP), is present at high concentrations in urine. THP can form polymers that entrap EVs during purification, reducing yield. Disruption of THP polymer networks with dithiothreitol (DTT) can release trapped EVs, but smaller THP fibres co-isolate with EVs during subsequent ultracentrifugation. To resolve these challenges, we describe here a dUC method that incorporates THP polymer reduction and alkaline washing to improve EV isolation and deplete both THP and other common protein contaminants. When applied to human expressed prostatic secretions in urine, we achieved relative enrichment of known prostate and prostate cancer-associated EV-resident proteins. Our approach provides a promising strategy for global proteomic analyses of urinary EVs. (hide) EV-METRIC 78% (97th 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 prostate cancer Focus vesicles extracellular vesicle 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 Filtration Protein markers EV: TSG101/ CD9 non-EV: Calnexin/ Tamm-Horsfall protein Proteomics yes Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods/Biomarker/New methodological development Sample Species Homo sapiens Sample Type Urine 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) 120 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 175000 Wash: volume per pellet (ml) 12.5 Wash: time (min) 130 Wash: Rotor Type SW 40 Ti Wash: speed (g) 175000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per 1E10 particles: 1.16 Western Blot Detected EV-associated proteins CD9/ TSG101 Detected contaminants Tamm-Horsfall protein Not detected contaminants Calnexin Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 145.9 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.20E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV210199
species	Homo sapiens
sample type	Urine
condition	prostate cancer
separation protocol	(d)(U)C Filtration
Exp. nr.	1
EV-METRIC %	78