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In this work we present droplet barcode sequencing for protein analysis (DBS\u2010Pro) to profile surface proteins on individual sEVs, facilitating identification of sEV\u2010subtypes within and between samples. The method allows for analysis of multiple proteins through use of DNA barcoded affinity reagents and sequencing as readout. High throughput single vesicle profiling is enabled through compartmentalization of individual sEVs in emulsion droplets followed by droplet barcoding through PCR. In this proof\u2010of\u2010concept study we demonstrate that DBS\u2010Pro allows for analysis of single sEVs, with a mixing rate below 2%. A total of over 120,000 individual sEVs obtained from a NSCLC cell line and from malignant pleural effusion (MPE) fluid of NSCLC patients have been analyzed based on their surface proteins. We also show that the method enables single vesicle surface protein profiling and by extension characterization of sEV\u2010subtypes, which is essential to identify the cellular origin of vesicles in heterogenous samples.<\/jats:p>","DOI":"10.1002\/jev2.12277","type":"journal-article","created":{"date-parts":[[2022,11,4]],"date-time":"2022-11-04T01:04:24Z","timestamp":1667523864000},"source":"Crossref","is-referenced-by-count":45,"title":["Characterizing single extracellular vesicles by droplet barcode sequencing for protein analysis"],"prefix":"10.1002","volume":"11","author":[{"given":"Mahsan","family":"Banijamali","sequence":"first","affiliation":[{"name":"Royal Institute of Technology (KTH), School of Engineering Sciences in Chemistry, Biotechnology and Health, Department of Gene Technology Science for Life Laboratory  Solna Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pontus","family":"H\u00f6jer","sequence":"additional","affiliation":[{"name":"Royal 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AlbaNova University Center  Stockholm Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christiane","family":"Stiller","sequence":"additional","affiliation":[{"name":"Royal Institute of Technology (KTH), School of Engineering Sciences in Chemistry, Biotechnology and Health, Department of Protein Science AlbaNova University Center  Stockholm Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vitaliy O.","family":"Kaminskyy","sequence":"additional","affiliation":[{"name":"Department of Oncology\u2010Pathology Karolinska Institutet  Solna Sweden"},{"name":"Department of Physiology and Pharmacology Karolinska Institutet  Stockholm Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Simon","family":"Ekman","sequence":"additional","affiliation":[{"name":"Department of Oncology\u2010Pathology Karolinska Institutet  Solna Sweden"},{"name":"Theme Cancer, Medical Unit head and Neck, Lung, and Skin Tumors, Thoracic Oncology Center 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