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Updated: Jun 17, 2026
Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation
Published on: June 21, 2021
Shotgun redox proteomics in sub-proteomes trapped on functionalised beads: Identification of proteins targeted by
Wentao Hu1, Sara Tedesco, Brian McDonagh
1Proteomics Research Group, Department of Biochemistry, Environmental Research Institute, University College Cork, Cork, Ireland.
Abstract:
If reactive oxygen species (ROS) levels exceed antioxidant defences, oxidative stress occurs; a common response to environmental pollutants. Proteins absorb ∼70% of ROS, altering amino acid side-chains. Cys (-SH) oxidises to sulphenic (-SOH), sulphinic (-SO(2)H), cysteic (-SO(3)H) acids and disulphide bridges (-S-S-). Two-dimensional electrophoresis (2DE) under-selects certain protein categories (e.g. extreme pI, small proteins) so activated thiol sepharose (ATS) was used to select sub-proteomes of thiol-containing proteins in menadione-exposed Escherichia coli. ATS bound thiol-containing proteins (but not oxidised thiols) via mixed disulphides. Tryptic digestion of bead-bound proteins was followed by LC-tandem MS. Many proteins were identified in controls with significantly fewer in menadione-treated cells (e.g. chaperonins, transcription/translation-related and ribosomal proteins; aminoacyl tRNA synthetases and metabolic enzymes. Non-denaturing ATS capture (followed by reduction) demonstrated lower specific activities of key enzymes which is attributed to thiol oxidation. This method may be generally useful in ecotoxicology for identification of oxidative stress targets.
