RT Journal Article
SR Electronic
T1 Proximity-labeling chemoproteomics defines the subcellular cysteinome and inflammation-responsive mitochondrial redoxome
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2023.01.22.525042
DO 10.1101/2023.01.22.525042
A1 Tianyang Yan
A1 Ashley R. Julio
A1 Miranda Villaneuva
A1 Anthony E. Jones
A1 Andréa B. Ball
A1 Lisa M. Boatner
A1 Alexandra C. Turmon
A1 Stephanie L. Yen
A1 Heta S. Desai
A1 Ajit S. Divakaruni
A1 Keriann M. Backus
YR 2023
UL http://biorxiv.org/content/early/2023/01/22/2023.01.22.525042.abstract
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.