Thiol-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 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 intraorganelle
spatial resolution with significantly enriched proteins localized
in the mitochondrial inner membrane and mitochondrial matrix.