Figure 3.

Labeling of cysteines with (IP)IAA and streptavidin enrichment of mitochondrial proteins for (A) Cys-LoC and (B) Cys-LOx

(A) For Cys-LoC, cell lysates are treated with iodoacetamide alkyne (IAA) to alkylate all reduced cysteines. Mitochondrial proteins that were previously biotinylated by TurboID are then enriched on streptavidin resin. Oxidized cysteines are then reduced with DTT and capped with IAA on resin.

(B) For Cys-LOx, cell lysates are treated with light isopropyl iodoacetamide alkyne (L-IPIAA) to alkylate all reduced cysteines. Mitochondrial proteins that were previously biotinylated by TurboID are then enriched on streptavidin resin. Oxidized cysteines are then reduced with DTT and capped with heavy isopropyl iodoacetamide alkyne (H-IPIAA) on resin.

(C) Chemical structures of biotin, IAA, L-IPIAA, and H-IPIAA.