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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV130020	1/1	Homo sapiens	NAY	(d)(U)C IAF UF	Tauro BJ	2013	38%
Study summary Full title Two distinct populations of exosomes are released from LIM1863 colon carcinoma cell-derived organoids All authors Tauro BJ, Greening DW, Mathias RA, Mathivanan S, Ji H, Simpson RJ Journal Mol Cell Proteomics Abstract Exosomes are naturally occurring biological nanomembranous vesicles (?40 to 100 nm) of endocytic ori (show more...)Exosomes are naturally occurring biological nanomembranous vesicles (?40 to 100 nm) of endocytic origin that are released from diverse cell types into the extracellular space. They have pleiotropic functions such as antigen presentation and intercellular transfer of protein cargo, mRNA, microRNA, lipids, and oncogenic potential. Here we describe the isolation, via sequential immunocapture using anti-A33- and anti-EpCAM-coupled magnetic beads, of two distinct populations of exosomes released from organoids derived from human colon carcinoma cell line LIM1863. The exosome populations (A33-Exos and EpCAM-Exos) could not be distinguished via electron microscopy and contained stereotypical exosome markers such as TSG101, Alix, and HSP70. The salient finding of this study, revealed via gel-based LC-MS/MS, was the exclusive identification in EpCAM-Exos of the classical apical trafficking molecules CD63 (LAMP3), mucin 13 and the apical intestinal enzyme sucrase isomaltase and increased expression of dipeptidyl peptidase IV and the apically restricted pentaspan membrane glycoprotein prominin 1. In contrast, the A33-Exos preparation was enriched with basolateral trafficking molecules such as early endosome antigen 1, the Golgi membrane protein ADP-ribosylation factor, and clathrin. Our observations are consistent with EpCAM- and A33-Exos being released from the apical and basolateral surfaces, respectively, and the EpCAM-Exos proteome profile with widely published stereotypical exosomes. A proteome analysis of LIM1863-derived shed microvesicles (sMVs) was also performed in order to clearly distinguish A33- and EpCAM-Exos from sMVs. Intriguingly, several members of the MHC class I family of antigen presentation molecules were exclusively observed in A33-Exos, whereas neither MHC class I nor MHC class II molecules were observed via MS in EpCAM-Exos. Additionally, we report for the first time in any extracellular vesicle study the colocalization of EpCAM, claudin-7, and CD44 in EpCAM-Exos. Given that these molecules are known to complex together to promote tumor progression, further characterization of exosome subpopulations will enable a deeper understanding of their possible role in regulation of the tumor microenvironment. (hide) EV-METRIC 38% (79th 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 Cell culture supernatant 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 IAF UF Protein markers EV: Alix/ EpCAM/ TSG101 non-EV: Proteomics yes Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Immunoaffinity capture Selected surface protein(s) A-33, EpCAM Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ TSG101/ EpCAM ELISA Antibody details provided? No Detected EV-associated proteins EpCAM Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

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EV-TRACK ID	EV130020
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C IAF UF
Exp. nr.	1
EV-METRIC %	38