Quantification of oxidative posttranslational modifications of cysteine thiols of p21ras associated with redox modulation of activity using isotope-coded affinity tags and mass spectrometry

Mahadevan, Sethuraman; Clavreul, Nicolas; Huang, Hongjuan; McComb, Mark E.; Costello, Catherine E.; Cohen, Richard A.

doi:10.1016/j.freeradbiomed.2006.12.012

Cited by 59 publications

(51 citation statements)

References 26 publications

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“…MS/MS analysis of the peptide is used to identify the respective protein and, at the same time, the oxidation sensitive cysteine(s). Although Sethuraman and coworkers have previously used ICAT technology to determine relative changes in the availability of free protein thiols in two separate samples (13,15,16), the OxICAT method provides the absolute ratio of reduced to oxidized protein within a single sample. This makes OxICAT also ideally suited for analyzing changes in the redox status of proteins in cells exposed to stress conditions, because neither changes in protein expression nor protein stability influence the OxICAT result.…”

Section: Resultsmentioning

confidence: 99%

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

Leichert

Gehrke

Gudiseva

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

486

542

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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

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Section: Resultsmentioning

confidence: 99%

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

Leichert

Gehrke

Gudiseva

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

486

542

View full text Add to dashboard Cite

show abstract

“…In practice, labeling can be done in high pH buffers to maximize labeling of all thiols regardless of pKa. Studies with the recombinant proteins, creatine phosphokinase and p21ras [47,48], indicate that oxidants decrease labeling to a large extent at known reactive cysteines ( Figure 7B), whereas labeling of non-reactive cysteines may be unchanged despite the same exposure ( Figure 7C). In the case of p21ras, which was S-glutathiolated in the presence of ONOO -and GSH, less ICAT was bound to cysteines 118, 181, 184, and 186.…”

Section: Mass Spectrometry and Proteomic Identification Of Reactive Cmentioning

confidence: 99%

Thiol oxidation in signaling and response to stress: Detection and quantification of physiological and pathophysiological thiol modifications

Ying

Clavreul

Mahadevan

et al. 2007

Free Radical Biology and Medicine

Self Cite

198

153

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Cysteine thiol modifications are increasingly recognized to occur under both physiological and pathophysiological conditions, making their accurate detection, identification, and quantification of growing importance. Amongst free cysteines, the bulk of modifications occur on a subset of cysteines that are more reactive. These exist as thiolate anions at physiological pH because of their surrounding electrostatic environment. Reagents with iodoacetamide active groups can be used to selectively label these reactive thiols with a high degree of selectivity. Thiol adducts can be detected by the failure to label with iodoacetamide or other reagents; restoration of labeling by specific reducing agents (eg. ascorbate or glutaredoxin), can be used to detect reversible S-nitroso and S-glutathione adducts. These adducts also may be detected with radiolabels and antibodies. S-glutathiolation in response to physiological stimuli may be detected in cells and tissues with glutathione ester labeled with biotin. Mass spectrometry can identify thiol modifications with precision, and with isotope-coded affinity tags, used to quantify modification of specific thiols. Combinations of these methods increase sensitivity and specificity, and enable quantification and precise identification of thiol modifications that occur under physiological and pathological conditions.

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“…Cohen and co-workers (12,13) have tailored this technique to probe the redox status of protein cysteines and identified redox-sensitive cysteines in cardiac sarcoplasmic reticulum proteins including ion channels that may be important for modulating cardiac functions. Furthermore they have also elegantly mapped differential cysteine thiol redox sensitivities of p21 ras GTPase toward peroxynitrite and oxidized glutathione (14). More recently, Svensson and co-workers (9) have used a modified ICAT method to discover over 100 in vitro Trx reduction targets in plants in which Trx-induced changes in protein disulfides were quantified.…”

mentioning

confidence: 99%

Elucidation of Thioredoxin Target Protein Networks in Mouse

Liu

et al. 2009

Molecular & Cellular Proteomics

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Thioredoxin 1 (Trx1) is a key redox modulator that is functionally conserved across a wide range of species, including plants, bacteria, and mammals. Using a conserved CXXC motif, Trx1 catalyzes the reduction of cysteine disulfides and S-nitrosothiols. In contrast to small molecular reductants such as glutathione and cysteine that can reduce a wide range of oxidized proteins, Trx1 reduces only selected proteins via specific protein-protein interaction. Trx1 has been shown to regulate numerous signal transduction pathways, and its dysfunctions have been implicated in several diseases, including cancer, inflammation, and neurodegenerative and cardiovascular diseases. Identification of Trx1 target proteins may help to identify novel signaling mechanisms that are important for Trx1 antistress responses. In this study, we performed an ICAT proteomics study for the identification of Trx1 target proteins from the hearts of a cardiac specific Trx1-overexpressing transgenic mouse model (Tg-Trx1). Trx1-reduced proteins were distinguished from Trx1-induced proteins by comparison of the ICAT results with those obtained using a parallel iTRAQ (isobaric tags for relative and absolute quantitation) protein expression analysis. We were able to identify 78 putative Trx1 reductive sites in 55 proteins. Interestingly we identified a few protein functional networks that had not been shown previously to be regulated by Trx1, including the creatine-phosphocreatine shuttle, the mitochondrial permeability transition pore complex, and the cardiac contractile apparatus. The results presented here suggest that in addition to a general antioxidant function, Trx1 may be involved in the coordination of a wide array of cellular functions for maintaining proper cardiac energy dynamics and facilitating muscle contraction. Molecular & Cellular Proteomics 8:1674 -1687, 2009. Thioredoxins (Trxs)1 are a class of antioxidant proteins that mediate the reduction of specific disulfide bonds and Snitrosothiols within oxidized proteins. Trx and the NADPH-dependent thioredoxin reductase (TrxR) form a protein reductive system that plays essential roles in the clearance of elevated reactive oxygen species, the repair of oxidatively modified proteins, and the restoration of cellular redox homeostasis. Two isoforms of Trx have been widely studied: Trx1 is mostly found in the cytosol and nucleus, whereas Trx2 is mitochondrion-specific. Each Trx isoform is coupled with its own TrxR systems. Trx1 has been shown to promote cell growth and proliferation and to inhibit apoptosis by modulating both caspase-dependent and -independent pathways. Trx1 has also been demonstrated to be a potent regulator of a wide variety of transcription factors and other gene expression regulators by preserving the reductive states of specific cysteines within transiently oxidized proteins such as nuclear factor B (1), hypoxia inducible factor-1␣ (2), histone deacetylase 4 (3), and glucocorticoid receptor (4). A wide range of bioanalytical techniques has been applied to identify Trx1 ta...

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Quantification of oxidative posttranslational modifications of cysteine thiols of p21ras associated with redox modulation of activity using isotope-coded affinity tags and mass spectrometry

Cited by 59 publications

References 26 publications

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

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

Thiol oxidation in signaling and response to stress: Detection and quantification of physiological and pathophysiological thiol modifications

Elucidation of Thioredoxin Target Protein Networks in Mouse

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