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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
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
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
Mass spectrometric strategies for studying albumin covalent modifications induced by xenobiotics and endogenous electrophilic cytotoxic compounds
No full textProtein covalent modification is involved in the toxic mechanisms of electrophilic xenobiotics as well as of endogenous cytotoxic compounds such as reactive carbonyl species generated by oxidative stress. Protein covalent modification is irreversible and unrepairable and besides inducing a loss of protein conformation and of functional integrity, it can make the adducted proteins immunogenic and pro-inflammatory. Identification and characterization of covalent protein adducts represent an important aspect of pharmaceutical analysis, not only to predict idiosyncratic reactions but also to elucidate cytotoxic mechanism at cellular levels and to find novel biomarkers of electrophilic/oxidative stress.
Human serum albumin (HSA) is considered the main blood protein target of circulating electrophilic compounds and this is due not only to its relevant plasma concentration (≈ 0.6 mM) but also because the presence of several accessible nucleophilic sites such as Cys34, Lys199 and 525. For this reason, HSA protein adducts are searched by using different analytical techniques and among these, mass spectrometry has attained a central role because of the wealth of structural and molecular information that can be obtained. MS approach not only reveals the identity of the adducted protein but also clarifies the stoichiometry of reaction, the aminoacid site undergoing biotransformation, the reaction products and hence the mechanism of reaction. Such an approach has been applied in our laboratory to fully elucidate the HSA binding by amoxicillin (AX) and by 4-hydroxy-trans-2-nonenal (HNE) which is an endogenous electrophilic RCS generated by lipid-peroxidation.
The binding of HSA with amoxicillin was investigated since it is considered a key process for the allergic response of the drug. HSA binding by AX was firstly studied by a top-down MS approach and then the identification of the adducted HSA sites was carried out by a novel bottom-up approach based on a precursor ion approach. At the lowest AX concentration, we detected one main AX-HSA adduct involving residues Lys 190, 199 or 541, whereas higher AX concentrations elicited a more extensive modification. These findings provide novel tools and insight for the study of protein haptenation and the mechanisms involved in AX-elicited allergic reactions.
HNE is one of most abundant and toxic lipid-peroxidation derived compound, which reacts with HSA. In a previous study we fully identified the protein adduct in terms of stoichiometry, reaction kinetic and sites of adduction [1]. We now set-up a LC-ESI-MS/MS approach in order to quantitate the protein adduct. The method consists to digest the protein and quantitate the adducted peptides by using a triple quadrupole MS analyzer working in multiple reaction monitoring mode. The isotope dilution technique was used for quantitative analysis and using deuterium labelled HNE adducted peptides. The method was firstly validated and it is now applied to measure HNE-adducted albumin in the sera of patients as a novel biomarker of oxidative stress.
References
1. Aldini G et al.Albumin is the main nucleophilic target of human plasma: a protective role against pro-atherogenic electrophilic reactive carbonyl species? Chem Res Toxicol. 2008,21,824-35
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Lemon peel and Limoncello liqueur: A proteomic duet
No full textCombinatorial peptide ligand libraries (CPLLs) have been adopted for investigating the proteomes of lemon peels and pulp, of a home-made alcoholic infusion of peels and of a very popular Italian liqueur called “Limoncello”, stated to be an infusion of the flavedo (the outer, yellow skin of lemons). The aim of this study was not only to perform the deepest investigation so far of the lemon peel proteome but also to assess the genuineness of the commercial liqueur via a three-pronged attack. First, different extraction techniques have been used for the characterization of the peel (and additionally of the pulp) proteome, secondly a home-made infusion has been analysed and finally the proteome of the commercial drink was checked. The peel (the flavedo, not the underlying layer called albedo) proteome has been evaluated via prior capture with CPLLs at different pH values (2.2 and 7.2). Via mass spectrometry analysis of the recovered fractions, after elution of the captured populations in 4% boiling SDS, we could identify a total of 1011 unique gene products in the peel extracts and 674 in the pulp, 264 proteins in the home-made infusion and just 8 proteins (and protein fragments), together with 12 peptides, in one Italian Limoncello produced in the Sorrento Region, thus proving the genuineness of this product. On the contrary, cheaper Limoncellos were devoid of any protein/peptide, casting doubts on their production from vegetable extracts. This could be the starting point for investigating the genuineness and natural origin of commercial drinks in order to protect consumers from adulterated products
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