Thiol-Cleavable Biotin for Chemical and Enzymatic Biotinylation and Its Application to Mitochondrial TurboID Proteomics
| Autor: | Ashley M Frankenfield, Haorong Li, Ling Hao, Shiori Sekine, Ryan Houston |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Proteomics
Streptavidin Lysine Biotin Mitochondrion 010402 general chemistry 01 natural sciences Protein biotinylation Article Mitochondrial Proteins chemistry.chemical_compound Structural Biology Humans Biotinylation Sulfhydryl Compounds Inner mitochondrial membrane Spectroscopy chemistry.chemical_classification DNA ligase 010401 analytical chemistry Mitochondria 0104 chemical sciences HEK293 Cells Biochemistry chemistry HeLa Cells |
| Zdroj: | J Am Soc Mass Spectrom |
| ISSN: | 1879-1123 1044-0305 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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