Covalent modifications of albumin CYS34 as biomarker of oxidative stress

Protein carbonylation is an irreversible process, induced by oxidants and reactive carbonyl species (RCS), the latter deriving from lipid peroxidation and oxidation of reducing sugars. Protein carbonylation is a cause/effect factor in oxidative based diseases and also an established biomarker of oxidative stress. From an analytical point of view, protein carbonylation is largely determined by immunological and spectroscopic techniques, which suffer from the intrinsic limitation of providing a very general and unspecific index of carbonyl damage. Hence, it is of great importance to set-up an analytical strategy aimed to identify, characterize and quantitate protein carbonyls in order to have a specific and reliable biomarker of oxidative stress. Cys34 residue of human serum albumin (HSA) represents the main target of oxidation and carbonylation in human plasma and this is explained by considering the high plasma content of HSA (≈ 0.6 mM), as well as the high reactivity and accessibility of the free Cys residue.[1] Different mass spectrometric approaches have been set-up in our laboratory to map the covalently oxidative modifications of Cys34, in order to identify suitable markers of early systemic oxidative stress. By using a top-down MS approach we found that Cys34 undergoes the following main oxidative modifications: cysteinylation, oxidation to sulfenic, sulfinic and sulfonic acid derivatives and carbonylation by reacting with RCS. Cysteinylated HSA was then monitored in different physio-pathological conditions involving oxidative stress, such as aging, diabetes, metabolic syndrome and nephropathy. Furthermore, we recently described an MS strategy based on the precursor-ion scanning technique which is able to specifically detect unknown covalent modifications of the Cys34 residue.[2] We are now on a further MS approach based on nanoLC coupled to the Orbitrap as the mass analyzer. Cys34 covalent adducts and oxidized forms are searched in a database of predicted variable modifications on Cys residues. We recently employed this approach to rapidly identify the covalent modifications of Cys34 in albumin exposed to a whole-phase cigarette smoke extract. The covalent Michael adducts of Cys34 with ACR and crotonaldehyde were identified, as well as the sulfinic and sulfonic acid derivatives.[3] [1] Aldini G et al., Chem Res Toxicol. 2008. [2] Aldini G. et al., J. Mass Spec. 2008. [3] Colombo G et al., Antioxid Redox Signal. 2010.