Characterization of albumin carbonylation by a tandem ms-precursor ion approach

Several experimental evidences confirm that protein carbonylation induced by reactive carbonyl species (RCS) is involved in the pathogenesis of several oxidative based diseases, including atherosclerosis and diabetic related diseases. By using a proteomic approach, we previously found that human serum albumin (HSA) is the main nucleophilic target of human plasma, due to the presence of some reactive nucleophilic aminoacid residues. We here report the set-up of a liquid chromatography electrospray ionisation multi-stage mass spectrometry (LC-ESIMS/ MS) approach based on precursor-ion scanning, aimed to identify and characterize the carbonylated adducts on His146, a reactive nucleophilic site of HSA able to form Michael adducts with alfa,beta-unsaturated aldehydes. The first step consisted of identifying the product ions to be used in the precursor ion scan, relative to the His146 peptide generated by the enzymatic digestion. In particular, by considering the trypsin/chymotrypsin digested peptide containing His146 (HPYFYAPELLFFAK), the ions at m/z 625.2 (y5) and m/z 964.6 (y8) were selected as product ions since not containing the target nucleophilic residue. MS data were then analyzed by using an algorithm developed by us, permitting a rapid and specific identification of the precursor-ions attributed to native and modified peptides. In a following step, the identified precursor ions were selected for product ion scan analysis in order to identify and characterize the covalent modification. The method has been validated by using 4-hydroxy-trans-nonenal (HNE) as a RCS model. Human serum was incubated in the presence of HNE (10 nmoles/ml), and then HSA was isolated by affinity chromatography and digested by using trypsin/chymotrypsin. The approach identified two peaks at m/z 871.9 [M+2H]2+ and m/z 949.9 [M+2H]2+ relative to the HPYFYAPELLFFAK, the former attributed to the native peptide and the latter to the His146-HNE adduct, as confirmed by MS/MS. The method was then successfully applied for the identification and characterization of the His146 covalent adducts of HSA incubated with 4-hydroxy-hexenal, crotonaldehyde, nonenale and acrolein. The tandem MS approach here reported could be a powerful tool for the rapid identification and characterization of carbonylated albumin in different pathological conditions.