A novel high resolution MS approach for the screening of 4-hydroxy-trans-2-nonenal sequestering agents.
An in vitro high resolution mass spectrometry (MS) method was set-up to test the ability of compounds, mixtures and extracts to inhibit protein carbonylation induced by reactive carbonyl species (RCS). The method consists of incubating the protein target (ubiquitin) with 4-hydroxy-trans-2-nonenal (HNE) in the presence and absence of the tested compound. After 24h of incubation, the reaction is stopped and the protein is analyzed by high-resolution MS. The extent of protein carbonylation is determined by measuring the area of the +11 multicharged peak of the HNE adduct in respect to the native form. The method was validated by measuring the effect of well-known RCS sequestering agents, namely aminoguanidine, pyridoxamine, hydralazine and carnosine, yielding a good reproducibility and the possibility to be automatable. All the compounds were found to dose-dependently inhibit the protein carbonylation with the following order of potency carnosine≈hydralazine≫aminoguanidine>pyridoxamine, as determined by calculating the UC50 values, that is the concentration required to inhibit ubiquitin carbonylation by 50%. A good correlation was found with the results obtained by measuring HNE consumption using an HPLC method optimized by a mobile phase set at pH 7.4, in order to stabilize the eluted adducts. The MS approach was then applied to test the effect of two selected natural extracts on protein carbonylation, i.e. green coffee bean extract and procyanidins from Vitis vinifera. In summary, this paper reports a validated and highly reproducible MS method to test the ability of pure compounds as well as natural extracts to act as protein carbonylation inhibitors.
Department of Pharmaceutical Sciences, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milan, Italy. Electronic address: mara.colzani@unimi.it.
Mara
M
Colzani
Department of Pharmaceutical Sciences, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milan, Italy. Electronic address: angela.criscuolo@studenti.unimi.it.
Angela
A
Criscuolo
Department of Pharmaceutical Sciences, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milan, Italy. Electronic address: danilo.demaddis@unimi.it.
Danilo
D
De Maddis
Department of Pharmaceutical Sciences, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milan, Italy. Electronic address: davide.garzon@unimi.it.
Davide
D
Garzon
Division of Food Bioscience, College of Biomedical and Health Sciences, Konkuk University, Chungju-Si, Republic of Korea. Electronic address: kyeum@kku.ac.kr.
Kyung-Jin
KJ
Yeum
Department of Pharmaceutical Sciences, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milan, Italy. Electronic address: giulio.vistoli@unimi.it.
Giulio
G
Vistoli
Department of Pharmaceutical Sciences, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milan, Italy. Electronic address: marina.carini@unimi.it.
Marina
M
Carini
Department of Pharmaceutical Sciences, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milan, Italy. Electronic address: giancarlo.aldini@unimi.it.
Giancarlo
G
Aldini
D000447:Aldehydes / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods*; D050050:Protein Carbonylation / Q000187:drug effects; D064449:Sequestering Agents / Q000737:chemistry*; D025801:Ubiquitin / Q000737:chemistry
D000447:Aldehydes; D064449:Sequestering Agents; D025801:Ubiquitin; C027576:4-hydroxy-2-nonenal
4-hydroxy-trans-2-nonenal; Advanced lipoxidation end products; Mass spectrometry; Protein carbonylation; RCS sequestering agents; Reactive carbonyls species