Updated project metadata. Updated publication reference for PubMed record(s): 34282141. This study aimed to identify subpopulations of extracellular vesicles (EVs) secreted by HeLa cells, and determine markers which enable to identify them, and which indicate their endosomal or plasma membrane origin. We used CD63 and CD9 as markers of EVs, and followed their intracellular trafficking using the RUSH assay. We used mass spectrometry to identify specific or common proteins in immunoprecipitated GFP+ EVs from HeLa transfected with the RUSH CD63-GFP or CD9-GFP, after a short (3h) or a long (24h) trafficking time from the endoplasmic reticulum.