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

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 [ref], 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 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.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Cell Culture, Macrophage

DISEASE(S): Disease Free

SUBMITTER: Tianyang Yan  

LAB HEAD: Keriann Backus

PROVIDER: PXD039626 | Pride | 2024-08-10

REPOSITORIES: Pride