PT  - JOURNAL ARTICLE
AU  - Tianyang Yan
AU  - Ashley R. Julio
AU  - Miranda Villaneuva
AU  - Anthony E. Jones
AU  - Andréa B. Ball
AU  - Lisa M. Boatner
AU  - Alexandra C. Turmon
AU  - Stephanie L. Yen
AU  - Heta S. Desai
AU  - Ajit S. Divakaruni
AU  - Keriann M. Backus
TI  - Proximity-labeling chemoproteomics defines the subcellular cysteinome and inflammation-responsive mitochondrial redoxome
AID  - 10.1101/2023.01.22.525042
DP  - 2023 Jan 01
TA  - bioRxiv
PG  - 2023.01.22.525042
4099  - http://biorxiv.org/content/early/2023/01/22/2023.01.22.525042.short
4100  - http://biorxiv.org/content/early/2023/01/22/2023.01.22.525042.full
AB  - 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, together with unexpected non-organelle specific TurboID-catalyzed proximity labeling. This mislabeling was minimized through simultaneous depletion of both endogenous biotin and newly translated TurboID fusion protein. 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.Competing Interest StatementThe authors have declared no competing interest.