biostudies-literatureLi HNational Institute of Neurological Disorders and StrokeNINDS NIH HHS2358-2365https://www.ebi.ac.uk/biostudies/studies/S-EPMC8898397biostudies-literatureUnknown32(9)Protein biotinylation via chemical or enzymatic reactions is often coupled with streptavidin-based enrichment and on-bead digestion in numerous biological applications. However, the popular on-bead digestion method faces major challenges of streptavidin contamination, overwhelming signals from endogenous biotinylated proteins, the lost information on biotinylation sites, and limited sequence coverage of enriched proteins. Here, we explored thiol-cleavable biotin as an alternative approach to elute biotinylated proteins from streptavidin-coated beads for both chemical biotinylation and biotin ligase-based proximity labeling. All possible amino acid sites for biotinylation were thoroughly evaluated in addition to the primary lysine residue. We found that biotinylation at lysine residues notaJournal of the American Society for Mass SpectrometryThiol-Cleavable Biotin for Chemical and Enzymatic Biotinylation and Its Application to Mitochondrial TurboID Proteomics.PMC8898397R01NS121608R01 NS121608Li HSekine SHao LFrankenfield AMHouston RfalseThiol-Cleavable Biotin for Chemical and Enzymatic Biotinylation and Its Application to Mitochondrial TurboID Proteomics.Protein biotinylation via chemical or enzymatic reactions is often coupled with streptavidin-based enrichment and on-bead digestion in numerous biological applications. However, the popular on-bead digestion method faces major challenges of streptavidin contamination, overwhelming signals from endogenous biotinylated proteins, the lost information on biotinylation sites, and limited sequence coverage of enriched proteins. Here, we explored thiol-cleavable biotin as an alternative approach to elute biotinylated proteins from streptavidin-coated beads for both chemical biotinylation and biotin ligase-based proximity labeling. All possible amino acid sites for biotinylation were thoroughly evaluated in addition to the primary lysine residue. We found that biotinylation at lysine residues nota2021-01-01T00:00:00Z2021 Sep2025-04-05T13:08:02.207Z2025-04-05T13:08:02.207ZS-EPMC88983973390997110.1021/jasms.1c00079