MassIVE MSV000087256

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

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Description

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. [doi:10.25345/C53F8Z] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: cleavable biotin ; TurboID ; Mitochondrion ; Proximity labeling ; Streptavidin ; biotin

Contact

Principal Investigators:
(in alphabetical order) 		Ling Hao, The George Washington University, United States of America
Submitting User: haolab

Publications

Haorong Li, Ashley M. Frankenfield, Ryan Houston, Shiori Sekine, Ling Hao.
Thiol-Cleavable Biotin for Chemical and Enzymatic Biotinylation and Its Application to Mitochondrial TurboID Proteomics.
J. Am. Soc. Mass Spectrom. 2021, 32, 9, 2358–2365.

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