biostudies-literatureKirkemo LLChan Zuckerberg Biohub Investigator ProgramNational Cancer InstituteNCI NIH HHSNational Institute of General Medical SciencesHarry and Dianna Hind ProfessorshipNIGMS NIH HHSNational Science Foundatione73982https://www.ebi.ac.uk/biostudies/studies/S-EPMC8983049biostudies-literatureUnknown11Characterization of cell surface proteome differences between cancer and healthy cells is a valuable approach for the identification of novel diagnostic and therapeutic targets. However, selective sampling of surface proteins for proteomics requires large samples (>10e6 cells) and long labeling times. These limitations preclude analysis of material-limited biological samples or the capture of rapid surface proteomic changes. Here, we present two labeling approaches to tether exogenous peroxidases (APEX2 and HRP) directly to cells, enabling rapid, small-scale cell surface biotinylation without the need to engineer cells. We used a novel lipidated DNA-tethered APEX2 (DNA-APEX2), which upon addition to cells promoted cell agnostic membrane-proximal labeling. Alternatively, we employed horseraeLifeCell-surface tethered promiscuous biotinylators enable comparative small-scale surface proteomic analysis of human extracellular vesicles and cells.PMC8983049R35 GM122451U01 CA244452F31 CA247527R01CA2483231F31CA247527U01CA244452R01 CA2483231650113R35GM122451Blelloch RKirkemo LLYang JGlasgow JEByrnes JRElledge SKWells JAfalseCell-surface tethered promiscuous biotinylators enable comparative small-scale surface proteomic analysis of human extracellular vesicles and cells.Characterization of cell surface proteome differences between cancer and healthy cells is a valuable approach for the identification of novel diagnostic and therapeutic targets. However, selective sampling of surface proteins for proteomics requires large samples (>10e6 cells) and long labeling times. These limitations preclude analysis of material-limited biological samples or the capture of rapid surface proteomic changes. Here, we present two labeling approaches to tether exogenous peroxidases (APEX2 and HRP) directly to cells, enabling rapid, small-scale cell surface biotinylation without the need to engineer cells. We used a novel lipidated DNA-tethered APEX2 (DNA-APEX2), which upon addition to cells promoted cell agnostic membrane-proximal labeling. Alternatively, we employed horsera2022-01-01T00:00:00Z2022 Mar2026-05-31T01:04:14.913Z2025-04-04T11:31:49.19ZS-EPMC89830493525766310.7554/eLife.73982