196,307 research outputs found
Detection and quantification of protein carbonyl-quenching activities by high-resolution mass spectrometry
No full textReactive carbonyl species (RCS) are highly electrophilic compounds generated in the organism upon oxidative stress. These molecules are implicated in the pathogenesis and progression of different oxidative-based disorders, such as diabetes, fibrosis and Alzheimer’s disease. Detoxification of RCS by nucleophilic compounds able to form unreactive adducts (carbonyl quenching agents) represents a promising strategy to reduce RCS concentration and to prevent their spontaneous and detrimental reaction with nucleophilic moieties of DNA, lipids and proteins1.
We analyzed and compared the quenching ability of known carbonyl quenching agents including aminoguanidine, hydralazine, pyridoxamine and carnosine. Their ability to prevent protein carbonylation was evaluated by testing different RCS such as 4-hydroxy-trans-2-nonenal, methylglyoxal, glyoxal and malondialdehyde. The quenching ability was quantified by using an innovative approach based on high-resolution mass spectrometry and on ubiquitin, as model protein2.
An approach based on mass spectrometry was applied to identify and characterize the reaction products between RCS and the 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, while aminoguanidine was more active on methylglyoxal and glyoxal. Hydralazine efficiently quenched all reactive carbonyl species, while pyridoxamine was particularly active on malondialdehyde. Selectivity was evaluated by testing the reactivity of the tested compounds towards pyridoxal (an endogenous aldehyde); the results indicated that only carnosine and pyridoxamine are highly selective.
The reaction products between RCS and the different carbonyl quenchers were fully characterized by high-resolution mass spectrometry, leading to the elucidation of the quenching reaction mechanisms.
We then tested the quenching ability of complex mixtures, such as natural extracts. This revealed the ability of green coffee bean extract and procyanidins from Vitis vinifera to prevent protein carbonylation, thus demonstrating that the proposed analytical strategy can be used to test the ability of pure compounds as well as of natural extracts as carbonyl quenching agents.
References
(1) Aldini, G.; Vistoli, G.; Stefek, M.; Chondrogianni, N.; Grune, T.; Sereikaite, J.; Sadowska-Bartosz, I.; Bartosz, G. Molecular Strategies to Prevent, Inhibit and Degrade Advanced Glycoxidation and Advanced Lipoxidation End Products. Free Radic. Res. 2013.
(2) Colzani, M.; Criscuolo, A.; De Maddis, D.; Garzon, D.; Yeum, K.-J.; Vistoli, G.; Carini, M.; Aldini, G. A Novel High Resolution MS Approach for the Screening of 4-Hydroxy-Trans-2-Nonenal Sequestering Agents. J. Pharm. Biomed. Anal. 2014, 91, 108–118
High-resolution mass spectrometry for the screening and characterization of protein carbonyl-quenching activities
No full textIntroduction
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. 201
Un Epitrapezio per Alessandro? Osservazioni sul “piccolo grande” Ercole della Cassetta Farnese di Capodimonte
No full textThe Gibbs Phenomenon for Multiple Fourier Integrals
No full textAbstractRestrict a smooth function to a domain bounded by a smooth surface. We study the summability of the Fourier integral of this function at points near the boundary of the domain
PROTEOMICS BASED DRUG DISCOVERY AND CHEMOPROTEOMICS
No full textGlobal proteome and post-translational modification profiling have been mainly applied to biomarker discovery, target identification and validatio
Mass spectrometric approaches for the identification and quantification of reactive carbonyl species protein adducts
No full textOur current knowledge of the occurrence of proteins covalently modified by reactive carbonyl species (RCS) generated by lipid peroxidation indicates their involvement as pathogenic factors associated with several chronic degenerative diseases. Proteomics and mass spectrometry (MS) in the last decade have played a fundamental role in this context, allowing the demonstration of the formation of RCS-protein adducts in vitro and in vivo under different experimental conditions. In conjunction with functional and computational studies, MS has been widely applied in vitro to study the stoichiometry of the protein-RCS adduct formation, and, by identifying the site(s) of modification, to elucidate the molecular mechanisms of protein carbonylation and the physiologic impact of such modification on protein function. This review will provide an update of the MS methods commonly used in detecting and characterizing protein modification by RCS generated by lipid peroxidation, among which 4-hydroxy-trans-2-nonenal and acrolein represent the most studied and cytotoxic compounds. Research in this field, employing state-of-the-art MS, is rapidly and continuously evolving, owing also to the development of suitable derivatization and enrichment procedures enabling the improve MS detectability of RCS-protein adducts in complex biological matrices. By considering the emerging role of RCS in several human diseases, unequivocal analytical approaches by MS are needed to provide levels of intermediate diagnostic biomarkers for human diseases. This review focuses also on the different MS-based approaches so far developed for RCS-protein adduct quantification. This article is part of a Special Issue entitled: Protein Modifications
Life for industrial sites End of Life
No full textIn the last decade, the decline in industrial settlements has emerged as an increasingly relevant and complex phenomenon, unveiling the limitations of the management models that are traditionally applied to these situations. This crucial problem is here addressed by adopting a conceptual framework, built to exploit a systemic perspective that moves from the recognition of the centrality of the company that used to inhabit the site. Specifically, operational guidelines are proposed and tested through their application to the case of enel’s power plant in Porto Tolle, being part of the Futur-e project. The results show how the conceptual model enables the identification of site-specific guidelines for the End Of Life (eol) strategy definition, and for the generation of opportunities for the entire area. If more traditional approaches were used, these opportunities would have remained undiscovered
Bochner-Riesz means of functions in weak-Lp
No full textThe Bochner-Riesz means of order δ≥0 for suitable test functions on RN are defined via the Fourier transform by {Mathematical expression}. We show that the means of the critical index {Mathematical expression}, do not map Lp,∞(RN) into Lp,∞(RN), but they map radial functions of Lp,∞(RN) into Lp,∞(RN). Moreover, if f is radial and in the Lp,∞(RN) closure of test functions, SRδf(x) converges, as R→+∞, to f(x) in norm and for almost every x in RN. We also observe that the means of the function |x|-N/p, which belongs to Lp,∞(RN) but not to the closure of test functions, converge for no x
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