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Hu, WT,Tedesco, S,McDonagh, B,Sheehan, D
Marine Environmental Research
Shotgun redox proteomics in sub-proteomes trapped on functionalised beads Identification of proteins targeted by oxidative stress
Optional Fields
Carbonylation Cysteine Oxidative stress Shotgun proteomics Escherichia coli CHEMISTRY
If reactive oxygen species (ROS) levels exceed antioxidant defences oxidative stress occurs a common response to environmental pollutants Proteins absorb similar to 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 duo! oxidation This method may be generally useful in ecotoxicology for identification of oxidative stress targets (C) 2009 Elsevier Ltd All rights reserved
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