Edaravone inhibits protein carbonylation by a direct carbonyl-scavenging mechanism: focus on reactivity, selectivity, and reaction mechanisms.

The aim of this study was to evaluate the ability of the well-known radical scavenging compound edaravone (EDA) to entrap and detoxify reactive carbonyl species (RCS) derived from lipid peroxidation [4-hydroxy-trans-2-nonenal (HNE), acrolein and glyoxal], as well as its ability to prevent RCS-induced protein carbonylation, by using hemoglobin (Hb) modified by HNE as an in vitro model. Through a combined HPLC and high-resolution mass spectrometric approach, we confirmed the ability of EDA to scavenge precursors for either advanced glycation or lipoxidation end products (EAGLEs), such as glyoxal, and demonstrated for the first time that EDA is also a potent quencher of alpha,beta-unsaturated aldehydes (providing mass spectral characterization of the adducts), being significantly more active than a series of well-known RCS sequestering agents. Direct infusion analysis of the intact protein and nano LC-ESI-MS/MS analysis of the tryptic digest, carried out on an LTQ-Orbitrap hybrid mass spectrometer, were used to study the modifications occurring on Hb after exposure to increasing HNE concentrations, providing evidence for Cys93 (Hb beta-chain) involvement in covalent attachment, and to demonstrate the ability of EDA dose-dependently to inhibit Hb carbonylation.

HNE Michael adducts to histidine and histidine-containing peptides as biomarkers of lipid-derived carbonyl stress in urines: LC-MS/MS profiling in Zucker obese rats.

A new liquid chromatography-tandem mass spectrometric (LC-MS/MS) approach, based on the precursor ion scanning technique using a triple-stage quadrupole, has been developed to detect free and protein-bound histidine (His) residues modified by reactive carbonyl species (RCS) generated by lipid peroxidation. This approach has been applied to urines from Zucker obese rats, a nondiabetic animal model characterized by obesity and hyperlipidemia, where RCS formation plays a key role in the development of renal and cardiac dysfunction. The immonium ion of His at m/z 110 was used as a specific product ion of His-containing peptides to generate precursor ion spectra, followed by MS2 acquisitions of each precursor ion of interest for structural characterization.