Mass spectroscopic characterization of protein modification by malondialdehyde.
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.
Ishii
Takeshi
T
Graduate School of Bioagricultural Sciences, Nagoya University, Chikusaku, Nagoya 464-8601, Japan.
Kumazawa
Shigenori
S
Sakurai
Toyo
T
Nakayama
Tsutomu
T
Uchida
Koji
K
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D018604:Epitope Mapping; D007328:Insulin / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D008239:Lysine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000276:immunology; D008315:Malondialdehyde / Q000032:analysis / Q000737:chemistry* / Q000276:immunology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D010449:Peptide Mapping; D011506:Proteins / Q000032:analysis / Q000737:chemistry* / Q000276:immunology; D012697:Serine Endopeptidases; D021241:Spectrometry, Mass, Electrospray Ionization
D000911:Antibodies, Monoclonal; D007328:Insulin; D010446:Peptide Fragments; D011506:Proteins; D008315:Malondialdehyde; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D008239:Lysine