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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140021	1/1	Homo sapiens	NAY	(d)(U)C	Amorim M	2014	33%
Study summary Full title The overexpression of a single oncogene (ERBB2/HER2) alters the proteomic landscape of extracellular vesicles All authors Amorim M, Fernandes G, Oliveira P, Martins-de-Souza D, Dias-Neto E, Nunes D Journal Proteomics Abstract ERBB2/HER2 amplification activates signaling cascades that lead to a tumor cell phenotype. However, (show more...)ERBB2/HER2 amplification activates signaling cascades that lead to a tumor cell phenotype. However, despite its remarkable importance in oncology, the consequences of HER2 amplification over the extracellular vesicles (EVs) content have not yet been investigated. Here, we isolated EVs secreted by HB4a, a mammary luminal epithelial cell line and C5.2, its HER2-overexpressing clone. We isolated two EV sets (20 and 100 K) by ultracentrifugation and used electron microscopy and nanoparticle tracking analysis for their morphological characterization. We employed GeLC-MS/MS combined with isotope-coded protein labeling to evaluate cell-derived proteins and LC-MS/MS label free spectral counting to quantify the EVs proteome. We found higher HER2 levels in both C5.2-derived EVs when compared with C5.2 cells, suggesting its preferential shuttling. Proteins capable of inducing malignant transformation are enriched in both C5.2 EV subsets, including two HER2-related proteins involved in cell motility and invasion, cofilin and CD44. MetaCore™ analysis indicated an enrichment of cell adhesion and cytoskeleton-remodeling pathways in C5.2 EVs, as well as proteins related to HER2 signaling, such as sphingosine-1-phosphate pathway. Together, our data indicate that in terms of protein content, distinct vesicle sets reinforce and complement each other. Our results also suggest that HER2-upregulated proteins from EVs may be relevant for cellular malignancy and can be potential biomarkers for HER2(+) cancer patients. (hide) EV-METRIC 33% (75th 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 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 Protein markers EV: HSP70/ Rab27B/ Flotilin1 non-EV: Proteomics yes Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Flotilin1/ HSP70/ Rab27B ELISA Antibody details provided? No Detected EV-associated proteins Rab27B Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

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EV-TRACK ID	EV140021
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C
Exp. nr.	1
EV-METRIC %	33