Mass spectrometric evidence for the existence of distinct modifications of different proteins by 2(E),4(E)-decadienal

Abstract

2(E),4(E)-Decadienal (DDE), a lipid peroxidation product, was found to covalently modify Lys residues of different proteins by different reactions using mass spectrometry (MALDI-TOF-MS and LC-ESI-MS). DDE mainly formed Lys Schiff base adducts with cytochrome c and ribonuclease A at 10 min, but these reversibly formed adducts almost disappeared after 24 h. In contrast, beta-lactoglobulin (beta-LG) was highly modified by DDE after 24 h. In addition to the Lys Schiff base adducts, DDE formed novel Lys pyridinium adducts as well as Cys Michael adducts with beta-LG.

Figure 1

MALDI-TOF mass spectra of 0.25 mM cytochrome c (A) or RNase A (B) modified by 20 equivalents of DDE (5 mM) in pH 7.4 phosphate buffer at 37 °C for various times. The mass increment of 134 Da is due to the DDE–Lys Schiff base formation.

Figure 2

MALDI-TOF mass spectra of 0.25 mM of β-LG modified by 20 equivalents of DDE (5 mM) in pH 7.4 phosphate buffer at 37 °C for various times. The mass increments of 134, 152 and 268 Da are due to the DDE–Lys Schiff base, DDE–Cys Michael adduct and DDE–Lys pyridinium adduct, respectively. Arrows a, b and c indicate that the pyridinium adduct was gradually formed after 2 to 24 h on β-LG A chain.

Figure 3

MALDI-TOF mass spectra of 0.25 mM of Cys121-carbamidomethylated β-LG modified by 20 equivalents of DDE (5 mM) in pH 7.4 phosphate buffer at 37 °C at various times. The mass increments of 134 and 269 Da are attributed to the DDE–Lys Schiff base and DDE–Lys pyridinium adduct, respectively.

Figure 4

MALDI-TOF mass spectra of chymotryptic digests of 0.25 mM of β-LG control (A) and β-LG modified by 20 equivalents of DDE (5 mM) after 10 min (B) or 24 h (C). The peaks marked with an asterisk are peptides modified by DDE through Lys pyridinium formation.

Figure 5

Characteristic fragment ions Py⁺ and M–285 of DDE–Lys pyridinium adducts.

Figure 6

Tandem mass spectra of modified peptides KVL (m/z 627.32), KIDAL (m/z 827.34), KGLDIQ (m/z 941.41) and DIQKVAGTW (m/z 1285.49) through DDE–Lys pyridinium formation. Fragment ions Py⁺ and M–285 are shown in Figure 5. Asterisks denote the modified residue.

Scheme 1. Proposed Reaction Mechanism for Formation of DDE–Lys Pyridinium Adducts 1a and 1b

Scheme 2. Reduction of DDE-Lys Pyridinium Adduct 1a by NaBH₄