Chemical Probes for Redox Signaling and Oxidative Stress

Abstract

Significance: Cellular reactive oxygen species (ROS) mediate redox signaling cascades that are critical to numerous physiological and pathological processes. Analytical methods to monitor cellular ROS levels and proteomic platforms to identify oxidative post-translational modifications (PTMs) of proteins are critical to understanding the triggers and consequences of redox signaling. Recent Advances: The prevalence and significance of redox signaling has recently been illuminated through the use of chemical probes that allow for sensitive detection of cellular ROS levels and proteomic dissection of oxidative PTMs directly in living cells.

Critical issues: In this review, we provide a comprehensive overview of chemical probes that are available for monitoring ROS and oxidative PTMs, and we highlight the advantages and limitations of these methods.

Future directions: Despite significant advances in chemical probes, the low levels of cellular ROS and low stoichiometry of oxidative PTMs present challenges for accurately measuring the extent and dynamics of ROS generation and redox signaling. Further improvements in sensitivity and ability to spatially and temporally control readouts are essential to fully illuminate cellular redox signaling.

Keywords: cysteine oxidation; oxidative stress; redox proteomics; redox sensors.

FIG. 1.

ROS and oxidative PTMs of cysteine. (A) ROS are derived from molecular oxygen through excitation and reduction. (B) Oxidative PTMs of cysteine highlighted in this review. PTMs, post-translational modifications; ROS, reactive oxygen species. Color images are available online.

FIG. 2.

Chemical probes for superoxide. (A) EPR-based spin traps form stable radicals with superoxide. (B) HE and MitoHE react with superoxide to form fluorescent products. (C) Lucigenin releases a photon on reaction with superoxide. EPR, electron paramagnetic resonance; HE, hydroethidine. Color images are available online.

FIG. 3.

Fluorescent probes for H₂O₂. (A) Boronate-protected fluorophores are deprotected by H₂O₂. (B) The benzil group is cleaved by H₂O₂ to form highly fluorescent 5-carboxyfluorescein. (C) HyPer enables ratiometric fluorescent measurements of H₂O₂ in situ. (D) Orp1 forms a disulfide bond on reaction with H₂O₂, which is relayed to the fused roGFP2, leading to a fluorescence change. H₂O₂, hydrogen peroxide; roGFP2, redox-sensitive green fluorescent protein 2. Color images are available online.

FIG. 4.

Chemical-proteomic platforms for general cysteine PTMs. (A) Structures of ICAT reagents. (B) OxICAT is a modified ICAT technology for oxidative cysteine PTMs. (C) A quantitative proteomic platform, isoTOP-ABPP, enables comprehensive identification and quantification of cysteine PTMs. (D) Caged electrophilic probes are developed to achieve in situ labeling of cysteine and analysis of cysteine PTMs within a physiological context. CBK1, caged α-bromomethylketone alkyne; IAA, idoacetamide alkyne; ICAT, isotope-coded affinity tags; isoTOP-ABPP, isotopic tandem orthogonal proteolysis activity-based protein profiling; LC-MS/MS, liquid chromatography-tandem mass spectrometry; OxICAT, oxidative isotope-coded affinity tag; TCEP, tris(2-carboxyethyl)phosphine; UV, ultraviolet. Color images are available online.

FIG. 5.

Chemical probes for sulfenic acids and sulfinic acids. (A) The reaction between sulfenic acids and dimedone. (B) Structures of dimedone-based probes. (C) A strained alkyne-based probe, BCN, can be conjugated to sulfenic acids. (D) The reaction between sulfinic acids and 2-nitroso benzoic acid derivatives. (E) NO-Bio is a nitroso-based chemical probe for sulfinic acids. BCN, bicycle[6.1.0]nonyne; DAz, azide-tagged dimedone; DYn, alkyne-tagged dimedone; NO-Bio, biotinylated 2-nitroso terepththalic acid. Color images are available online.

FIG. 6.

Chemical probes for S-glutathionylation. (A) Structures of biotinylated glutathione derivatives, BioGSH and BioGEE. (B) Glycine surrogates with bioorthogonal handles for metabolic incorporation. (C) The glycine surrogates can be incorporated into GSH catalyzed by mutated GSs in living cells, which enables proteomic analyses of native glutathionylation. BioGEE, biotinylated glutathione ethyl ester; BioGSH, biotinylated GSH; CuAAC, copper(I)-catalyzed alkyne-azide cycloaddition; GS, glutathione synthetase; GSH, reduced glutathione. Color images are available online.