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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140189	1/1	Homo sapiens	Urine	(d)(U)C UF	Liu X	2014	25%
Study summary Full title Intraluminal proteome and peptidome of human urinary extracellular vesicles All authors Liu X, Chinello C, Musante L, Cazzaniga M, Tataruch D, Calzaferri G, James Smith A, De Sio G, Magni F, Zou H, Holthofer H Journal Proteomics Clin Appl Abstract PURPOSE: Urinary extracellular vesicles (UEVs) are a novel source for disease biomarker discovery. H (show more...)PURPOSE: Urinary extracellular vesicles (UEVs) are a novel source for disease biomarker discovery. However, Tamm-Horsfall protein (THP) is still a challenge for proteomic analysis since it can inhibit detection of low-abundance proteins. Here, we introduce a new approach that does not involve an ultracentrifugation step to enrich vesicles and that reduces the amount of THP to manageable levels. EXPERIMENTAL DESIGN: UEVs were dialyzed and ultrafiltered after reduction and alkylation. The retained fraction was digested with trypsin to reduce the remaining THP and incubated with deoxycholate (DOC). The internal peptidome and internal proteome were analyzed by LC-ESI-MS. RESULTS: A total of 942 different proteins and 3115 unique endogenous peptide fragments deriving from 973 different protein isoforms were identified. Around 82% of the key endosomal sorting complex required for transport components of UEVs generation could be detected from the intraluminal content. CONCLUSIONS AND CLINICAL RELEVANCE: Our UEVs preparation protocol provides a simplified way to investigate the intraluminal proteome and peptidome, in particular the subpopulation of UEVs of the trypsin-resistant class of exosomes (positive for tumor susceptibility gene101) and eliminates the majority of interfering proteins such as THP. This method allows the possibility to study endoproteome and endopeptidome of UEVs, thus greatly facilitating biomarker discovery. (hide) EV-METRIC 25% (56th 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 extracellular 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 UF Protein markers EV: TSG101 non-EV: Tamm-Horsfall glycoprotein Proteomics yes 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 Pelleting performed No Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins TSG101 Detected contaminants Tamm-Horsfall glycoprotein Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

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EV-TRACK ID	EV140189
species	Homo sapiens
sample type	Urine
condition	NAY
separation protocol	(d)(U)C UF
Exp. nr.	1
EV-METRIC %	25