Thiol-cleavable Biotin for Chemical and Enzymatic Biotinylation and its Application to Mitochondrial TurboID Proteomics

ABSTRACT: Protein biotinylation via chemical or enzymatic reactions is often coupled with streptavidin-based enrichment and on-beads digestion in numerous biological applications. However, the popular on-beads digestion method faces major challenges of streptavidin contamination, the lost information of 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 besides the primary lysine residue. We found that biotinylation at lysine residues notably reduces the trypsin digestion efficiency which can be mitigated by the thiol-cleavable biotinylation method. We then evaluated the applicability of thiol-cleavable biotin as a substrate for proximity labeling in living cells, where TurboID biotin ligase was engineered onto the mitochondrial inner membrane facing the mitochondrial matrix. As a proof-of-principle study, thiol-cleavable biotin-assisted TurboID proteomics achieved remarkable intra-organelle spatial resolution with significantly enriched proteins localized in the mitochondrial inner membrane and mitochondrial matrix.

INSTRUMENT(S): Q-Exactive HFX

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Ling Hao  

PROVIDER: MSV000087256 | MassIVE | Wed Apr 21 09:12:00 BST 2021

REPOSITORIES: MassIVE