Mass spectrometric characterization of covalent modification of human serum albumin by 4-hydroxy-trans-2-nonenal

Several pieces of evidence indicate that albumin modified by HNE is a promising biomarker of systemic oxidative stress and that HNE-modified albumin may contribute to the immune reactions triggered by lipid peroxidn.-derived antigens. In this study, we found by HPLC anal. that HNE is rapidly quenched by human serum albumin (HSA) because of the covalent adduction to the different accessible nucleophilic residues of the protein, as demonstrated by electrospray ionization mass spectrometry (ESI-MS) direct infusion expts. (one to nine HNE adducts, depending on the molar ratio used, from 1:0.25 to 1:5 HSA: HNE). An LC-ESI-MS/MS approach was then applied to enzymically digested HNE-modified albumin, which permitted the identification of 11 different HNE adducts, 8 Michael adducts (MA) and 3 Schiff bases (SB), involving nine nucleophilic sites, namely: His67 (MA), His146 (MA), His242 (MA), His288 (MA), His510 (MA), Lys 195 (SB), Lys 199 (MA, SB), Lys525 (MA, SB) and Cys34 (MA). The most reactive HNE-adduction site was found to be Cys34 (MA) followed by Lys199, which primarily reacts through the formation of a Schiff base, and His146, giving the corresponding HNE Michael adduct. These albumin modifications are suitable tags of HNE-adducted albumin and could be useful biomarkers of oxidative and carbonylation damage in humans