Protein 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 . 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.
Mass spectrometric strategies for studying albumin covalent modifications induced by xenobiotics and endogenous electrophilic cytotoxic compounds / D. Garzon, M. Colzani, L. Cannizzaro, M. Carini, G. Aldini. ((Intervento presentato al 7. convegno Nuove prospettive in Chimica Farmaceutica tenutosi a Savigliano nel 2013.
|Titolo:||Mass spectrometric strategies for studying albumin covalent modifications induced by xenobiotics and endogenous electrophilic cytotoxic compounds|
GARZON, DAVIDE (Primo)
COLZANI, MARA (Secondo)
CARINI, MARINA (Penultimo)
ALDINI, GIANCARLO (Ultimo)
|Data di pubblicazione:||29-mag-2013|
|Settore Scientifico Disciplinare:||Settore CHIM/08 - Chimica Farmaceutica|
|Enti collegati al convegno:||Società Chimica Italiana|
|Citazione:||Mass spectrometric strategies for studying albumin covalent modifications induced by xenobiotics and endogenous electrophilic cytotoxic compounds / D. Garzon, M. Colzani, L. Cannizzaro, M. Carini, G. Aldini. ((Intervento presentato al 7. convegno Nuove prospettive in Chimica Farmaceutica tenutosi a Savigliano nel 2013.|
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