Quantifying changes in the thiol redox proteome upon oxidative stress in vivo

Abstract

Antimicrobial levels of reactive oxygen species (ROS) are produced by the mammalian host defense to kill invading bacteria and limit bacterial colonization. One main in vivo target of ROS is the thiol group of proteins. We have developed a quantitative thiol trapping technique termed OxICAT to identify physiologically important target proteins of hydrogen peroxide (H 2O2) and hypochlorite (NaOCl) stress in vivo. OxICAT allows the precise quantification of oxidative thiol modifications in hundreds of different proteins in a single experiment. It also identifies the affected proteins and defines their redox-sensitive cysteine(s). Using this technique, we identified a group of Escherichia coli proteins with significantly (30 -90%) oxidatively modified thiol groups, which appear to be specifically sensitive to either H 2O2 or NaOCl stress. These results indicate that individual oxidants target distinct proteins in vivo. Conditionally essential E. coli genes encode one-third of redoxsensitive proteins, a finding that might explain the bacteriostatic effect of oxidative stress treatment. We identified a select group of redox-regulated proteins, which protect E. coli against oxidative stress conditions. These experiments illustrate that OxICAT, which can be used in a variety of different cell types and organisms, is a powerful tool to identify, quantify, and monitor oxidative thiol modifications in vivo.chaperone ͉ proteomics ͉ thiol modification

The biphasic redox sensing of SENP3 accounts for the HIF-1 transcriptional activity shift by oxidative stress

“…Oxidative modification of protein thiols is now considered as an emerging role in cell signaling. It has been well recognized that the thiol oxidation state of reactive cysteine residues in proteins controls the function of the proteins and the pathways that they are part of [49][50][51][52][53][54] . We hypothesize that, during the biphasic redox sensing, the different cysteines of a given protein molecule might be sequentially oxidized by different levels of ROS, thus leading to different conformational characteristics and functional statuses of the protein.…”

Radical-free biology of oxidative stress

“…New discovery-based redox proteomic methods with mass spectrometry use Isotope-Coded Affinity Tag (ICAT) reagent to label thiols with heavy ( 13 C) and light ( 12 C) forms of biotin-labeled iodoacetamide to quantify redox states of specific Cys residues in proteins (56,108). Sequential addition of the heavy isotopomer before reduction and then addition of light isotopomer after reduction provides a measure of the reduced fraction of specific Cys residues in terms of the ratio of labeling.…”

Interplay between redox and protein homeostasis

“…By calculating the ratio Ox/Red peptides, the redox state of protein populations can be determined. 99 …”

“…Peptides that initially contained reduced thiols would yield mass peaks corresponding to peptides labeled with light ICAT and initially oxidized peptides would yield a higher mass peak, corresponding to peptides labeled with heavy ICAT. 99 The thiol groups on proteins are a major target of ROS and play an important role in redox signaling, so the absolute ratio of reduced to oxidized proteins in a sample is extremely relevant and can be obtained with the OxICAT method. This feature makes OxICAT properly tailored for determining any change in the redox state of proteins that were exposed to stress conditions, allowing exhaustive identification of all oxidized cysteine residues, and precise determination of the oxidized cysteine residues within polypeptides in one single analysis.…”

“…98 Leichert and colleagues used an isotope-coded affinity tag (ICAT), a shotgun proteomic strategy approach, combined with a thiol trapping method to develop a novel quantitative technique, termed OxICAT. 99 Apart from the cleavable biotin affinity tag, the ICAT reagent comprises an iodoacetamide element and a 9-carbon linker, with isotope light 12 C-and heavy 13 C-form. 100 After acidic quenching, oxidized versus reduced cysteine residues are differentially labeled with the heavy and light ICAT reagents.…”