SICyLIA-cTMT enables dissecting redox proteome dynamics with high accuracy and depth at micrograms scale (PART 6 = Metabolically stressed liver tissues without carrier).

ABSTRACT: Cysteine oxidative modifications are fundamental signaling events that regulate cellular functions under both physiological and pathological conditions. However, our understanding of these modifications is limited by their low abundance and the technical challenges associated with studying their dynamics in a comprehensive and unbiased manner. Here, we developed the SICyLIA-TMT workflow, which uses light and heavy iodoacetamide (IAA) to sequentially label reduced and reversibly oxidized cysteine residues within the same sample. By applying tandem mass tags (TMTs) to differentially label peptides of each experimental conditions, our strategy allows simultaneous analysis of both oxidative modification dynamics and protein levels across multiple experimental conditions from only micrograms of starting material. Furthermore, to enhance the detection of low-abundance, cysteine residues carrying oxidative modifications, we dedicated a TMT channel as carrier for heavy IAA-labelled peptides (SICyLIA-cTMT). This strategy remarkably increased the quantification of reversibly oxidized cysteine residues and enabled precise stoichiometry calculations. Last, combining SICyLIA-TMT with the latest generation of MS instruments, Orbitrap Astral, we halved the measuring time in the mass spectrometer without impacting the depth of the redox proteome. We showed the applicability of SICyLIA-TMT to both cells in culture and full organs under stress condition. Hence, SICyLIA-cTMT is a versatile method that achieves unparalleled quantification accuracy and depth of the redox proteome while significantly reducing MS analysis time, making a significant step change in the study of oxidative signaling.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Liver

DISEASE(S): Obesity

SUBMITTER: Sergio Lilla  

LAB HEAD: Sara Rossana Zanivan

PROVIDER: PXD061865 | Pride | 2025-10-01

REPOSITORIES: Pride