Oxidative protein labeling in mass-spectrometry-based proteomics

Oxidation of proteins and peptides is a common phenomenon, and can be employed as a labeling technique for mass-spectrometry-based proteomics. Nonspecific oxidative labeling methods can modify almost any amino acid residue in a protein or only surface-exposed regions. Specific agents may label reactive functional groups in amino acids, primarily cysteine, methionine, tyrosine, and tryptophan. Nonspecific radical intermediates (reactive oxygen, nitrogen, or halogen species) can be produced by chemical, photochemical, electrochemical, or enzymatic methods. More targeted oxidation can be achieved by chemical reagents but also by direct electrochemical oxidation, which opens the way to instrumental labeling methods. Oxidative labeling of amino acids in the context of liquid chromatography(LC)–mass spectrometry (MS) based proteomics allows for differential LC separation, improved MS ionization, and label-specific fragmentation and detection. Oxidation of proteins can create new reactive groups which are useful for secondary, more conventional derivatization reactions with, e.g., fluorescent labels. This review summarizes reactions of oxidizing agents with peptides and proteins, the corresponding methodologies and instrumentation, and the major, innovative applications of oxidative protein labeling described in selected literature from the last decade

Monitoring Intracellular Redox Changes in Ozone-Exposed Airway Epithelial Cells

Background: The toxicity of many xenobiotic compounds is believed to involve oxidative injury to cells. Direct assessment of mechanistic events involved in xenobiotic-induced oxidative stress is not easily achievable. Development of genetically encoded probes designed for monitoring intracellular redox changes represents a methodological advance with potential applications in toxicological studies. Objective: We tested the utility of redox-sensitive green fluorescent protein (roGFP)–based redox sensors for monitoring real-time intracellular redox changes induced by xenobiotics in toxicological studies. Methods: roGFP2, a reporter of the glutathione redox potential (E(GSH)), was used to monitor E(GSH) in cultured human airway epithelial cells (BEAS-2B cells) undergoing exposure to 0.15–1.0 ppm ozone (O(3)). Cells were imaged in real time using a custom-built O(3) exposure system coupled to a confocal microscope. Results: O(3) exposure induced a dose- and time-dependent increase of the cytosolic E(GSH). Additional experiments confirmed that roGFP2 is not directly oxidized, but properly equilibrates with the glutathione redox couple: Inhibition of endogenous glutaredoxin 1 (Grx1) disrupted roGFP2 responses to O(3), and a Grx1-roGFP2 fusion protein responded more rapidly to O(3) exposure. Selenite-induced up-regulation of GPx (glutathione peroxidase) expression–enhanced roGFP2 responsiveness to O(3), suggesting that (hydro)peroxides are intermediates linking O(3) exposure to glutathione oxidation. Conclusion: Exposure to O(3) induces a profound increase in the cytosolic E(GSH) of airway epithelial cells that is indicative of an oxidant-dependent impairment of glutathione redox homeostasis. These studies demonstrate the utility of using genetically encoded redox reporters in making reliable assessments of cells undergoing exposure to xenobiotics with strong oxidizing properties