SICyLIA-cTMT dissects redox proteome dynamics with high accuracy and depth at microgram scale.

Cysteine oxidative modifications are critical signaling events regulating cellular functions, but their low abundance and dynamic nature pose technical challenges. We developed the SICyLIA-TMT workflow, which sequentially labels reduced and reversibly oxidized cysteines with light and heavy iodoacetamide (IAA) within the same sample. The inclusion of tandem mass tags (TMTs) enables simultaneous quantification of oxidative modification dynamics and protein levels across multiple conditions using micrograms of material. To improve the detection of low-abundance oxidized cysteines, a dedicated TMT channel serves as a carrier for heavy IAA-labeled peptides (SICyLIA-cTMT), enhancing quantification and enabling precise stoichiometry calculations. We demonstrate the workflow's applicability to cultured cells and full organs under stress. SICyLIA-cTMT achieves unprecedented depth and accuracy in redox proteome analysis while reducing mass spectrometry time. Combining SICyLIA-TMT with latest mass spectrometry technologies further halves the acquisition time without compromising coverage, improving throughput and enabling comprehensive studies of oxidative signaling.