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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV120044	1/1	Homo sapiens	"Cerebrospinal fluid"	(d)(U)C DG	Street JM	2012	44%
Study summary Full title Identification and proteomic profiling of exosomes in human cerebrospinal fluid All authors Street JM, Barran PE, Mackay CL, Weidt S, Balmforth C, Walsh TS, Chalmers RT, Webb DJ, Dear JW Journal J Transl Med Abstract BACKGROUND: Exosomes are released from multiple cell types, contain protein and RNA species, and hav (show more...)BACKGROUND: Exosomes are released from multiple cell types, contain protein and RNA species, and have been exploited as a novel reservoir for disease biomarker discovery. They can transfer information between cells and may cause pathology, for example, a role for exosomes has been proposed in the pathophysiology of Alzheimer's disease. Although studied in several biofluids, exosomes have not been extensively studied in the cerebrospinal fluid (CSF) from humans. The objective of this study was to determine: 1) whether human CSF contains exosomes and 2) the variability in exosomal protein content across individuals. METHODS: CSF was collected from 5 study participants undergoing thoraco-abdominal aortic aneurysm repair (around 200 - 500 ml per participant) and low-density membrane vesicles were concentrated by ultracentrifugation. The presence of exosomes was determined by western blot for marker proteins, isopycnic centrifugation on a sucrose step gradient and transmission electron microscopy with immuno-labelling. Whole protein profiling was performed using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR). RESULTS: Flotillin 1 and tumor susceptibility gene 101 (TSG101), two exosomal marker proteins, were identified in the ultracentrifugation pellet using western blot. These markers localized to a density consistent with exosomes following isopycnic centrifugation. Transmission electron microscopy visualized structures consistent with exosomes in size and appearance that labelled positive for flotillin 1. Therefore, the pellet that resulted from ultracentrifugation of human CSF contained exosomes. FT-ICR profiling of this pellet was performed and 84-161 ions were detected per study participant. Around one third of these ions were only present in a single study participant and one third were detected in all five. With regard to ion quantity, the median coefficient of variation was 81% for ions detected in two or more samples. CONCLUSIONS: Exosomes were identified in human CSF and their proteome is a potential new reservoir for biomarker discovery in neurological disorders such as Alzheimer's disease. However, techniques used to concentrate exosomes from CSF need refinement to reduce variability. In this study we used relatively large starting volumes of human CSF, future studies will focus on exosome isolation from smaller real life clinical samples; a key challenge in the development of exosomes as translational tools. (hide) EV-METRIC 44% (96th 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 "Cerebrospinal fluid" 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 non-EV: Proteomics yes EV density (g/ml) 1.1-1.14 Show all info Study aim Omics Sample Species Homo sapiens Sample Type "Cerebrospinal fluid" 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) 60 Density gradient Lowest density fraction 0.25 Highest density fraction 2 Orientation Top-down Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Flotilin1/ TSG101 Characterization: Particle analysis EM EM-type transmission EM/ immune EM EM protein Flotillin1 Image type Close-up, Wide-field

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EV-TRACK ID	EV120044
species	Homo sapiens
sample type	Cerebrospinal fluid
condition	NAY
separation protocol	(d)(U)C DG
Exp. nr.	1
EV-METRIC %	44