biostudies-literatureShin SInstitute for Basic ScienceNational Research Foundation of Korea (NRF)National Research Foundation of KoreaInstitute for Basic Science (IBS)554https://www.ebi.ac.uk/biostudies/studies/S-EPMC11082246biostudies-literatureUnknown7(1)Promiscuous labeling enzymes, such as APEX2 or TurboID, are commonly used in in situ biotinylation studies of subcellular proteomes or protein-protein interactions. Although the conventional approach of enriching biotinylated proteins is widely implemented, in-depth identification of specific biotinylation sites remains challenging, and current approaches are technically demanding with low yields. A novel method to systematically identify specific biotinylation sites for LC-MS analysis followed by proximity labeling showed excellent performance compared with that of related approaches in terms of identification depth with high enrichment power. The systematic identification of biotinylation sites enabled a simpler and more efficient experimental design to identify subcellular localized proCommunications biologySuper-resolution proximity labeling with enhanced direct identification of biotinylation sites.PMC11082246IBS-R008-D1NRF-2022R1A2B5B03001658NRF-2019M3E5D3073104NRF-2021R1A6A3A01087055NRF-2020R1C1C1013927Shin SJang DGLee SYKim JKang MGRhee HWKim JSfalseSuper-resolution proximity labeling with enhanced direct identification of biotinylation sites.Promiscuous labeling enzymes, such as APEX2 or TurboID, are commonly used in in situ biotinylation studies of subcellular proteomes or protein-protein interactions. Although the conventional approach of enriching biotinylated proteins is widely implemented, in-depth identification of specific biotinylation sites remains challenging, and current approaches are technically demanding with low yields. A novel method to systematically identify specific biotinylation sites for LC-MS analysis followed by proximity labeling showed excellent performance compared with that of related approaches in terms of identification depth with high enrichment power. The systematic identification of biotinylation sites enabled a simpler and more efficient experimental design to identify subcellular localized pro2024-01-01T00:00:00Z2024 May2026-07-14T20:31:07.546Z2025-08-12T03:05:00.653ZS-EPMC110822463872455910.1038/s42003-024-06112-w