Mass Spectroscopic Characterization of Protein Modification by Malondialdehyde

Abstract

Malondialdehyde (MDA), a naturally occurring dialdehyde produced in the membrane by lipid peroxidation, is a strong alkylating agent of primary amino groups. We recently raised a monoclonal antibody (mAb1F83) directed to the lipofuscin-like MDA--lysine adduct and demonstrated the presence of immunoreactivity to the antibody in the atherosclerotic lesions, in which intense positivity was associated primarily with macrophage-derived foam cells (Yamada et al., (2001) J. Lipid Res. 42, 1187-1196). To identify the structure of the epitope in the protein recognized by mAb1F83, in the present study, we exposed chain B from bovine insulin (insulin B chain) to MDA and characterized the MDA adducts by mass spectrometry. The MDA-modified insulin B chain was digested with V8 protease, and the resulting peptides were subjected to liquid chromatography--electrospray ionization--mass spectrometry (LC--ESI--MS/MS). The MS/MS analyses confirmed the formation of N-propenal- (+54 Da) and dihydropyridine-type (DHP, +134 Da) adducts in both Lys29 and the N-terminus of insulin B chain. The ELISA analysis of HPLC fractions of peptides, including the DHP adducts using mAb1F83, showed that the immunoreactivity of the DHP--lysine adduct was more significant than the DHP--N-terminus adduct. The results of this study chemically characterized that the MDA adducts such as DHP-type adducts generated in the epsilon-amino group of lysine and N-terminal amino acid residues in the protein and the structure of the epitope recognized by mAb1F83 were DHP--lysine adducts in protein.

Recognition of Malondialdehyde-modified Proteins by the C Terminus of Complement Factor H Is Mediated via the Polyanion Binding Site and Impaired by Mutations Found in Atypical Hemolytic Uremic Syndrome

“…When proteins are treated with MDA, N-propenal (Fig. 1) is the major adduct with very little complex structures formed (37,43,44). If MDHDC is desired in abundance, extra acetaldehyde is needed in modifications (10).…”

“…MDA Modifications-MDA modifications were performed mainly as described previously (35,37). Briefly, BSA (2 mg/ml) was incubated in 100 mM phosphate (pH 7.2) with 1-85 mM MDA in rotation at 37°C for 17 h to produce MDA-BSA.…”

Modifying Apolipoprotein A-I by Malondialdehyde, but Not by an Array of Other Reactive Carbonyls, Blocks Cholesterol Efflux by the ABCA1 Pathway

“…Loss of precursor peptide was calculated using the ratio of the peak area of precursor peptide of apoA-I from control or modified apoA-I to that of the corresponding 15 N-labeled peptide from 15 N-labeled apoA-I. We focused on Lys residues first, because previous studies identified them as major targets of MDA (24,48). When apoA-I was exposed to a 20-fold molar ratio of MDA for 24 h, isotope dilution revealed that multiple peptides containing one or more Lys residues were lost in high yield.…”

Reactive Carbonyls Are a Major Th2-Inducing Damage-Associated Molecular Pattern Generated by Oxidative Stress

“…Similar to GA, 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA) are among the most common bioactive aldehyde products of oxidative stress (17), whose adducts have been implicated in various immune pathologies (5,18) and linked to induction of adaptive immunity (19,20). Because HNE and MDA attack on proteins can result in RC adduction (21,22), we investigated the significance of such structures and confirm here that RCs are indeed the central immune-active moieties via which these aldehydes modulate Ag immunogenicity. Furthermore, we show that RCs induce a profound Th2 biasing effect regardless of their mode of generation.…”