A quantitative thiol reactivity profiling platform to analyze redox and electrophile reactive cysteine proteomes

Fu, Ling; Li, Zongmin; Liu, Keke; Tian, Caiping; He, Ji-Xiang; He, Jingjing; He, Fuchu; Xu, Ping; Yang, Jing

doi:10.1038/s41596-020-0352-2

Cited by 56 publications

(58 citation statements)

References 71 publications

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“…Application of this strategy in mammalian cells reveals that heightened reactivity indeed serves as a good predictor of cysteine functionality 22 , even though some intrinsically reactive and functional cysteines might be inaccessible to the probe due to chemical or steric reasons. Based on the same principle, we recently developed a chemoproteomic method named quantitative thiol reactivity profiling (QTRP), and systematically profiled cysteine reactivity in Drosophila and mammalian cells 23,24 .…”

Section: Resultsmentioning

confidence: 99%

“…However, we noticed that our database did not include the peroxidatic (or catalytic) cysteines in 2-Cys peroxiredoxins (e.g., PRDX-2) that are well known to be redox-reactive 30 . To maintain the intrinsic reactivity of profiled cysteines, we employed native lysis conditions 23 , under which the peroxidatic cysteine in PRDX-2 was predominantly disulfide- linked and therefore would not have been labeled and detected ( Supplementary Fig. 3).…”

Section: Resultsmentioning

confidence: 99%

“…For intrinsic cysteine reactivity, protein lysates were separated into two identical aliquots and incubated with either 10 μM or 100 μM IPM at room temperature (RT,~25°C) for 1 h with rotation and light protection. For SH labeling, protein lysates were incubated with 100 μM IPM at RT for 1 h with rotation and light protection 23 . For -SOH, protein lysates were incubated with 5 mM BTD at 37°C for 1 h with rotation 29 .…”

Section: Methodsmentioning

confidence: 99%

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Global profiling of distinct cysteine redox forms reveals wide-ranging redox regulation in C. elegans

Jin

Liu

et al. 2021

Nat Commun

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Post-translational changes in the redox state of cysteine residues can rapidly and reversibly alter protein functions, thereby modulating biological processes. The nematode C. elegans is an ideal model organism for studying cysteine-mediated redox signaling at a network level. Here we present a comprehensive, quantitative, and site-specific profile of the intrinsic reactivity of the cysteinome in wild-type C. elegans. We also describe a global characterization of the C. elegans redoxome in which we measured changes in three major cysteine redox forms after H2O2 treatment. Our data revealed redox-sensitive events in translation, growth signaling, and stress response pathways, and identified redox-regulated cysteines that are important for signaling through the p38 MAP kinase (MAPK) pathway. Our in-depth proteomic dataset provides a molecular basis for understanding redox signaling in vivo, and will serve as a valuable and rich resource for the field of redox biology.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Global profiling of distinct cysteine redox forms reveals wide-ranging redox regulation in C. elegans

Jin

Liu

et al. 2021

Nat Commun

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“…This observation probably does not exclude S-PRDXylation. Widespread S-PRDXylation could also be in accordance with the identification of many oxidation-sensitive cysteines in redox proteomics studies, as these would not distinguish S-PRDXylation from other intra-or intermolecular disulfides [28]. Besides a role as a post-translational modification impacting the function of specific proteins, widespread peroxiredoxinylation could in principle also serve as a redox buffer.…”

Section: Discussionmentioning

confidence: 99%

The Human 2-Cys Peroxiredoxins form Widespread, Cysteine-Dependent- and Isoform-Specific Protein-Protein Interactions

et al. 2021

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Redox signaling is controlled by the reversible oxidation of cysteine thiols, a post-translational modification triggered by H2O2 acting as a second messenger. However, H2O2 actually reacts poorly with most cysteine thiols and it is not clear how H2O2 discriminates between cysteines to trigger appropriate signaling cascades in the presence of dedicated H2O2 scavengers like peroxiredoxins (PRDXs). It was recently suggested that peroxiredoxins act as peroxidases and facilitate H2O2-dependent oxidation of redox-regulated proteins via disulfide exchange reactions. It is unknown how the peroxiredoxin-based relay model achieves the selective substrate targeting required for adequate cellular signaling. Using a systematic mass-spectrometry-based approach to identify cysteine-dependent interactors of the five human 2-Cys peroxiredoxins, we show that all five human 2-Cys peroxiredoxins can form disulfide-dependent heterodimers with a large set of proteins. Each isoform displays a preference for a subset of disulfide-dependent binding partners, and we explore isoform-specific properties that might underlie this preference. We provide evidence that peroxiredoxin-based redox relays can proceed via two distinct molecular mechanisms. Altogether, our results support the theory that peroxiredoxins could play a role in providing not only reactivity but also selectivity in the transduction of peroxide signals to generate complex cellular signaling responses.

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“…Finally, to use probes for competitive residue-specific proteomics, their relative intensity between the light and the heavy channel needs to be quantified. For this purpose, complex in-house software 3,34 or workarounds in existing software 4 had to be used, so far. Therefore, we extended the quantification tool IonQuant 8 -another FragPipe component -to allow quantification of modified peptides in MSFragger Offset Searches as well as in conventional Closed Searches, in which the modified residue is specified.…”

Section: Resultsmentioning

confidence: 99%

Profiling the Proteome-Wide Selectivity of Diverse Electrophiles

Zanon¹,

Yu²,

Zhang³

et al. 2021

Preprint

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<p><a>Targeted covalent inhibitors are powerful entities in drug discovery, but their application has so far mainly been limited to addressing cysteine residues. The development of cysteine-directed covalent inhibitors has largely profited from determining their proteome-wide selectivity using competitive residue-specific proteomics. Several probes have recently been described to monitor other amino acids using this technology and many more electrophiles exist to modify proteins. Nevertheless, a direct, proteome‑wide comparison of the selectivity of diverse probes is still entirely missing. Here, we developed a completely unbiased workflow to analyse electrophile selectivity proteome‑wide and applied it to directly compare 54 alkyne probes containing diverse reactive groups. In this way, we verified and newly identified probes to monitor a total of nine different amino acids as well as the <i>N</i>‑terminus proteome‑wide. This selection includes the first probes to globally monitor tryptophans, histidines and arginines as well as novel tailored probes for methionines, aspartates and glutamates.</a></p>

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A quantitative thiol reactivity profiling platform to analyze redox and electrophile reactive cysteine proteomes

Cited by 56 publications

References 71 publications

Global profiling of distinct cysteine redox forms reveals wide-ranging redox regulation in C. elegans

Global profiling of distinct cysteine redox forms reveals wide-ranging redox regulation in C. elegans

The Human 2-Cys Peroxiredoxins form Widespread, Cysteine-Dependent- and Isoform-Specific Protein-Protein Interactions

Profiling the Proteome-Wide Selectivity of Diverse Electrophiles

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