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. 2011 Dec 2;286(48):41893-41903.
doi: 10.1074/jbc.M111.296236. Epub 2011 Oct 5.

Using quantitative redox proteomics to dissect the yeast redoxome

Nicolas Brandes  1 , Dana Reichmann  1 , Heather Tienson  1 , Lars I Leichert  1 , Ursula Jakob  2
Affiliations

Using quantitative redox proteomics to dissect the yeast redoxome

Nicolas Brandes et al. J Biol Chem. .

Abstract

To understand and eventually predict the effects of changing redox conditions and oxidant levels on the physiology of an organism, it is essential to gain knowledge about its redoxome: the proteins whose activities are controlled by the oxidation status of their cysteine thiols. Here, we applied the quantitative redox proteomic method OxICAT to Saccharomyces cerevisiae and determined the in vivo thiol oxidation status of almost 300 different yeast proteins distributed among various cellular compartments. We found that a substantial number of cytosolic and mitochondrial proteins are partially oxidized during exponential growth. Our results suggest that prevailing redox conditions constantly control central cellular pathways by fine-tuning oxidation status and hence activity of these proteins. Treatment with sublethal H(2)O(2) concentrations caused a subset of 41 proteins to undergo substantial thiol modifications, thereby affecting a variety of different cellular pathways, many of which are directly or indirectly involved in increasing oxidative stress resistance. Classification of the identified protein thiols according to their steady-state oxidation levels and sensitivity to peroxide treatment revealed that redox sensitivity of protein thiols does not predict peroxide sensitivity. Our studies provide experimental evidence that the ability of protein thiols to react to changing peroxide levels is likely governed by both thermodynamic and kinetic parameters, making predicting thiol modifications challenging and de novo identification of peroxide sensitive protein thiols indispensable.

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Figures

FIGURE 1.
FIGURE 1.
Organelle-specific protein enrichment for enhanced peptide identification in acid-trapped cell lysates. Organelle-specific proteins were enriched by organelle isolation, fully reduced, and labeled with either light or heavy ICAT reagent. Then, light- and heavy-labeled peptides were mixed in a 1:1 ratio, and LC-MS/MS was conducted on each organelle sample. A graphical representation of LC/MS runs of ICAT-labeled mitochondrial, nuclear, and vacuolar peptides in the range of m/z = 2275–2525 Da is shown (left panels). In each panel, up to six different peptides that were identified by MS/MS analysis are indicated by colored boxes. For representative purposes, one peptide per organelle was selected and the respective mass spectrum is shown. The two mass peaks correspond to the light and heavy ICAT-labeled peptides mixed in a 1:1 ratio. The right panel shows the corresponding m/z range of total yeast cell lysate after differential thiol trapping using OxICAT. The organelle-specific peptides were identified within the total cell lysate by their m/z value and position in the HPLC run and are highlighted. The corresponding mass spectra indicate their in vivo redox status in exponentially growing yeast cells.
FIGURE 2.
FIGURE 2.
The redox baseline, localization, and oxidation states of yeast proteins under non-stress conditions. Identified yeast proteins (392 total) were plotted according to their predominant cellular localization based on annotations in the S. cerevisiae Genome Database (A) and their in vivo oxidation status during exponential growth as determined by OxICAT (B; see supplemental Table S1). Most nuclear proteins are assigned both cytoplasmic and nuclear localizations and were thus grouped together. All other proteins that were found in more than one compartment are grouped under multiple locations.
FIGURE 3.
FIGURE 3.
Peroxide-sensitive yeast proteins: subcellular localization and affected pathways. A, relative cellular distribution of the 41 identified H2O2-sensitive yeast proteins as compared with the relative cellular distribution of all 392 identified yeast proteins. B, peroxide-sensitive yeast proteins grouped according to their cellular functions (see Table 2).
FIGURE 4.
FIGURE 4.
Comparison of structural properties between reduced (group I), peroxide-sensitive (group II), and oxidized (group III) cysteine thiols. A, pKa values of cysteine thiols classified as reduced, peroxide-sensitive, or oxidized as predicted by PROPKA (version 2.0). Distribution of pKa values is significantly different among the three sets according to Kruskal-Wallis one-way analysis of variance (p value = 0.01). The median of each group is shown by a solid line. B, distribution of interatomic interactions between the identified cysteine thiols and residues found within a 3 Å distance in reduced (black bars), peroxide sensitive (light gray bars), and oxidized (dark gray bars) groups. C, prediction of disulfide bond-forming propensity according to PROPKA 2.0, which uses a S–S distance criterion of 2.5 Å. D, relative accessibility of cysteine thiols. E, STRIDE analysis of secondary structure elements harboring the identified cysteines. Crystal structures of the respective proteins were used as input (see supplemental Table S2). pos., positively; neg. negatively.
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