Structural Analysis of Peptide‐Acetaldehyde Adducts by Mass Spectrometry and Production of Antibodies Directed Against Nonreduced Protein‐Acetaldehyde Adducts

Acetaldehyde can form protein‐acetaldehyde adducts (AAs) in vivo and may play a role in the genesis of alcoholic liver disease. The nature of the chemical modification of proteins by acetaldehyde in vivo has not been elucidated. In vitro, acetaldehyde can form reversible adducts including a Schiff's base with lysine (K) and imidazolidinone with terminal amino groups of proteins such as human hemoglobin (Hb). In this study, we used FAB/MS to analyze the products of peptide‐AAs (pep‐AAs) formed by incubating acetaldehyde with Hb peptides. We then used an octabranched multiple antigen peptide (MAP) system containing Hb peptide‐AAs to raise antibodies. Three Hb peptides [i.e., 8‐pep consisting of 8 residues (V1HLTPVEK8) at the N‐terminus of β‐chain of human sickle‐cell Hb, 11‐pep‐gly consisting of 11 residues (G56NPKVKAHGKK66) in a segment of β‐chain rich in lysine, and 11‐pep‐pro that consists of the same sequence as 11‐pep‐gly, except G56 was replaced by proline (P)] were incubated with 1 mM acetaldehyde at 4°C for 7d without NaCNBH3 (nonreduced conditions). Analysis by FAB/MS showed that 8‐pep formed an imidazolidinone at the N‐terminal valine, 11‐pep‐gly formed a Schiff's base and imidazolidinone at the N‐terminus, whereas 11‐pep‐pro that lacks a free α‐amino group formed only a Schiff's base at K59. By contrast, incubation of these Hb peptides with 250 mM acetaldehyde and NaCNBH3 at 37°C for 1 hr (reduced conditions) produced mono‐and diethylated modifications of all available K residues, as well as the N‐terminal amino group. MAP‐11‐pep‐gly and MAP‐11‐pep‐pro were prepared and reacted with 1 mM acetaldehyde under nonreduced conditions. Antibodies against these nonreduced MAP‐11 ‐pep‐AAs were raised. By ELISA assay, antibodies against nonreduced MAP‐11‐pep showed reactivity with Hb‐AA prepared under nonreduced conditions. Unexpectedly, these antibodies also reacted with Hb‐AA prepared under reduced conditions as well. Our studies therefore showed: (1) acetaldehyde can modify peptides with or without NaCNBH3 producing different chemical modifications; (2) lysine residues, although located in close vicinity in a peptide, are not equally reactive in forming a stable AA; and (3) antibodies raised by nonreduced MAP‐11 ‐pep‐AA immunogens can recognize both reduced and nonreduced epitopes in Hb‐AA.