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. 2008 Jun 17;105(24):8197-202.
doi: 10.1073/pnas.0707723105. Epub 2008 Feb 14.

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

Lars I Leichert  1 , Florian Gehrke, Harini V Gudiseva, Tom Blackwell, Marianne Ilbert, Angela K Walker, John R Strahler, Philip C Andrews, Ursula Jakob
Affiliations

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

Lars I Leichert et al. Proc Natl Acad Sci U S A. .

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(2)O(2)) 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(2)O(2) or NaOCl stress. These results indicate that individual oxidants target distinct proteins in vivo. Conditionally essential E. coli genes encode one-third of redox-sensitive 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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Determining the oxidation state of protein thiol using ICAT technology (OxICAT). A hypothetical cellular protein, which exists in either the reduced (Upper) or disulfide-linked form (Lower), is incubated under denaturing conditions to expose all of its cysteine side chains. In step I, isotopically light 12C- ICAT reagent (green ovals) is added, which irreversibly modifies all reduced cysteines in the protein. In step II, all oxidized cysteines are reduced with Tris(2-carboxyethyl)phosphine (TCEP) and subsequently modified with isotopically heavy 13C- ICAT reagent (red ovals). In step III, the protein mixture is digested and ICAT-labeled peptides are purified by using the biotin-affinity tag. In step IV, quantitative MS of the protein mixture reveals the extent of thiol modification in any given peptide. Peptide sequence and identity of the modified cysteine are determined by MS/MS.
Fig. 2.
Fig. 2.
Using OxICAT to visualize disulfide bond formation in vitro. Reduced, zinc-reconstituted Hsp33 (50 μM) is incubated in the presence of 2 mM H2O2 at 43°C. Samples are taken at specific time points during the oxidation process, and either TCA precipitated to stop all thiol-disulfide exchange reactions and subjected to the OxICAT method (A–C), or analyzed for chaperone activity (D). (A) Detail of the mass spectrum of OxICAT-treated reduced Hsp33. The two cysteines in peptide 232–236 are almost exclusively labeled with light ICAT (calculated m/z = 1023.4710). (B) Detail of the mass spectrum of OxICAT labeled Hsp33 after 60 min of activation. The two cysteines in peptide 232–236 are mostly labeled with heavy ICAT, leading to a mass shift of 18 Da (calculated m/z = 1041.5314). (C) The monoisotope peak corresponding to the Hsp33 peptide 232–236 in the reduced (red) (m/z = 1023.46), partially oxidized (int) (m/z = 1032.51), or fully oxidized (ox) (m/z = 1041.55) form was plotted against incubation time. (D) Influence of Hsp33 (0.3 μM) on the aggregation of chemically denatured citrate synthase (75 nM) at 30°C. The light scattering signal 4 min after addition of citrate synthase was plotted against incubation time. The light scattering signal of citrate synthase in the absence of Hsp33 was used as 0% chaperone activity, whereas the signal in the presence of fully active Hsp33 was set to 100%.
Fig. 3.
Fig. 3.
Visualization of OxICAT results. Graphical representation of the LC-MS analysis of aerobically growing E. coli cells. Intensity of each mass signal is given as fraction of blackness. Each mass signal corresponds to one OxICAT labeled peptide. Peptide 44–62 of ribosomal protein RpsQ elutes as ICAT pair with a mass difference of 9.0 Da. Mass signal with lower m/z has incorporated light ICAT, representing the reduced form of the peptide whereas mass signal with higher m/z has incorporated heavy ICAT and represents the oxidized form of the peptide. DppA 257–275 contains a cysteine that is part of a structural disulfide bond and elutes as single mass peak labeled with heavy ICAT.
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