Proximity-labeling chemoproteomics defines the subcellular cysteinome and inflammation-responsive mitochondrial redoxome

Yan, T.; Julio, A. R.; Villanueva, M.; Jones, A. E.; Ball, A. B.; Boatner, L. M.; Turmon, A. C.; Yen, S. L.; Desai, H. S.; Divakaruni, A. S.; Backus, K. M.

doi:10.1101/2023.01.22.525042

bioRxiv · 10.1101/2023.01.22.525042

Proximity-labeling chemoproteomics defines the subcellular cysteinome and inflammation-responsive mitochondrial redoxome

Yan, T.·Julio, A. R.·Villanueva, M.·Jones, A. E.·Ball, A. B.·Boatner, L. M.·Turmon, A. C.·Yen, S. L.·Desai, H. S.·Divakaruni, A. S.·Backus, K. M.

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Abstract

Proteinaceous cysteines function as essential sensors of cellular redox state. Consequently, defining the cysteine redoxome is a key challenge for functional proteomic studies. While proteome-wide inventories of cysteine oxidation state are readily achieved using established, widely adopted proteomic methods such as OxiCat, Biotin Switch, and SP3-Rox, they typically assay bulk proteomes and therefore fail to capture protein localization-dependent oxidative modifications. To obviate requirements for laborious biochemical fractionation, here, we develop and apply an unprecedented two step cysteine capture method to establish the Local Cysteine Capture (Cys-LoC), and Local Cysteine Oxidation (Cys-LOx) methods, which together yield compartment-specific cysteine capture and quantitation of cysteine oxidation state. Benchmarking of the Cys-LoC method across a panel of subcellular compartments revealed more than 3,500 cysteines not previously captured by whole cell proteomic analysis. Application of the Cys-LOx method to LPS stimulated murine immortalized bone marrow-derived macrophages (iBMDM), revealed previously unidentified mitochondria-specific inflammation-induced cysteine oxidative modifications including those associated with oxidative phosphorylation. These findings shed light on post-translational mechanisms regulating mitochondrial function during the cellular innate immune response.

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Yan, T., Julio, A. R., Villanueva, M., Jones, A. E., Ball, A. B., Boatner, L. M., Turmon, A. C., Yen, S. L., Desai, H. S., Divakaruni, A. S., Backus, K. M.. 2023-01-22. Proximity-labeling chemoproteomics defines the subcellular cysteinome and inflammation-responsive mitochondrial redoxome. https://doi.org/10.1101/2023.01.22.525042

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