Abstract
Protein carbonylation has been known as the major form of irreversible protein modifications and is also widely used as an indicator of oxidative stress in the biological environment. In the presence of oxidative stress, biological systems tend to produce large amounts of carbonyl moieties; these carbonyl groups do not have particular UV-Vis and fluorescence spectroscopic characteristics that we can differentiate, observe, and detect. Thus, their detection and quantification can only be performed using specific chemical probes. Commercially available fluorescent probes to detect specific carbonylation in biological systems have been used, but their chemical portfolio is still very limited. This protocol outlines the methods and procedures employed to synthesize a probe, (E,Z)-2-(2-(2-hydroxybenzylidene)hydrazonyl)-5-nitrophenol (2Hzin5NP), and assess its impact on carbonylation in human cells. The synthesis involves several steps, including the preparation of its hydrazone compounds mimicking cell carbonyls, 2-Hydrazinyl 5-nitrophenol, (E,Z)-2-(2-ethylidenehydrazonyl)-5-nitrophenol, and the final product (E,Z)-2-(2-(2-hydroxybenzylidene)hydrazonyl)-5-nitrophenol. The evaluation of fluorescence quantum yield and subsequent cell culture experiments are detailed for the investigation of 2Hzin5NP effects on cell proliferation and carbonylation. Key features • This protocol builds upon probe development using click chemistry method by Dilek et al. [1], and its biolabeling application in renal cancer cell lines. • The non-fluorescent probe has a fast reaction with carbonyl moieties at neutral pH to form a stable fluorescent product leading to a spectroscopic alteration. • Microscopic and fluorometric analyses can distinguish the exogenous and endogenous ROS-induced carbonylation profile in human dermal fibroblasts along with renal cell carcinoma. • Carbonylation level that differs in response to exogenous and endogenous stress in healthy and cancer cells can be detected by the newly synthesized fluorescent probe.