High-resolution mass spectrometry for the screening and characterization of protein carbonyl-quenching activities

Introduction Reactive carbonyl species (RCS) are highly electrophilic compounds generated in the organism upon oxidative stress and implicated in the pathogenesis/progression of different oxidative- based disorders, such as diabetes, fibrosis and Alzheimer’s disease. Carbonyl quenchers are nucleophilic compounds able to form unreactive adducts with RCS. Carbonyl quenching represents a promising strategy to reduce RCS concentration and to prevent their spontaneous and detrimental reaction with nucleophilic moieties of DNA, lipids and proteins [1]. In this work, we analyzed and compared the carbonyl quenching ability of different carbonyl quenchers and of natural extracts. Methods The in vitro quenching ability of carbonyl quenchers such as aminoguanidine, hydralazine, pyridoxamine and carnosine was tested on different RCS, including 4-hydroxy-trans-2-nonenal, methylglyoxal and malondialdehyde. The ability to prevent protein carbonylation was quantified by using an innovative approach based on high-resolution mass spectrometry and on ubiquitin, as model protein [2]. Results The different RCS formed specific adducts on distinct nucleophilic residues of ubiquitin. Increasing amounts of carbonyl quenchers prevented the formation of protein adducts, as determined by calculating the UC50 values - that is the concentration required to inhibit ubiquitin carbonylation by 50%. Quantitative analyses showed different carbonyl quenching activities: carnosine efficiently quenched the 4-hydroxy-trans-2-nonenal, aminoguanidine was more active on methylglyoxal, pyridoxamine was particularly active on malondialdehyde, while hydralazine efficiently quenched all RCS. The reactivity of the tested quenchers towards pyridoxal (as endogenous aldehyde) was tested to estimate their selectivity: carnosine and pyridoxamine were highly selective. The quenching ability of complex mixtures, such as natural extracts, was also tested, revealing the ability of green coffee bean extract and procyanidins from Vitis vinifera to prevent protein carbonylation. Conclusions The proposed analytical strategy was used to characterize and compare the carbonyl quenching ability of pure compounds and to detect the ability of natural extracts to prevent protein carbonylation. The analysis of the reaction products between RCS and carbonyl quenchers by high-resolution mass spectrometry led to the elucidation of the quenching mechanisms. Novel Aspect Our strategy, based on high resolution of mass spectrometry, represents an innovative platform to test the carbonyl quenching ability of pure compounds as well as of natural extracts. References [1] Aldini, G. et al. Molecular Strategies to Prevent, Inhibit and Degrade Advanced Glycoxidation and Advanced Lipoxidation End Products. Free Radic. Res. 2013 [2] Colzani, M. et al. Novel High Resolution MS Approach for the Screening of 4-Hydroxy-Trans-2-Nonenal Sequestering Agents. J. Pharm. Biomed. Anal. 2014