Summary- paper 23:
Proximity-labeling chemoproteomics defines the subcellular cysteinome and inflammation-responsive mitochondrial redoxome
Tianyang Yan, Ashley R. Julio, Miranda Villanueva, Anthony E. Jones, Andréa B. Ball, Lisa M. Boatner, Alexandra C. Turmon, Stephanie L. Yen, Heta S. Desai, Ajit S. Divakaruni, Keriann M. Backus
Cysteines in proteins are sensors of redox changes and many approches look at global snapshots of cystine oxidation states in specific conditions. However these approaches do not provide organelle specific redox responses.
ROS generation in mitochondria is especially high, and function of mitochondrial proteins is especially affected in pro-inflammatory programs in macrophages. What is the extent of mitochondrial cysteine oxidation in these states?
- TurboID proximity based biotinylation with cysteine redox state analysis to enable in situ subcellular cysteine fractionation and quantitative measurement of cysteine oxidation state: Local Cysteine Capture (Cys-LoC).
- Transiently overexpression of TurboID fusion proteins with localization sequences targeted to cytosol, endoplasmic reticulum, Golgi, mitochondrial, and nucleus.
- TurboID proximal proteins are first biotinylated in situ.
- Lysis and cysteine capping with the highly reactive iodoacetamide alkyne (IAA).
- Biotinylated proteins enriched on streptavidin resin and subjected to sequence specific proteolysis to release all IAA-tagged peptides derived from the TurboID modified proteins.
- Peptide-level click conjugation to biotin-azide followed by a second enrichment on neutravidin resin to capture of biotinylated cysteine peptides derived from TurboID modified proteins, followed by MS/MS.
See organelle variability in the efficiency of the Cys-LoC pipeline working- works well for nucleus and cytosol, less effective for ER/ Golgi/ mito. Increasing labeling time (to 1h) and adding exogenous biotin to 500 µM improves efficiency somewhat.
The biotinylation is promiscuous (observed by streptavidin-rhodamine), even though Mito-TurboID-eGFP reporter was mostly mitochondrial (observed by co-localization with mitotracker-tetramethylrhodamine ethyl ester (TMRE)).