Characterization of the deoxyguanosine-lysine cross-link of methylglyoxal

Abstract

Methylglyoxal is a mutagenic bis-electrophile that is produced endogenously from carbohydrate precursors. Methylglyoxal has been reported to induce DNA-protein cross-links (DPCs) in vitro and in cultured cells. Previous work suggests that these cross-links are formed between guanine and either lysine or cysteine side chains. However, the chemical nature of the methylglyoxal induced DPC have not been determined. We have examined the reaction of methylglyoxal, deoxyguanosine (dGuo), and Nα-acetyllysine (AcLys) and determined the structure of the cross-link to be the N2-ethyl-1-carboxamide with the lysine side chain amino group (1). The cross-link was identified by mass spectrometry and the structure confirmed by comparison to a synthetic sample. Further, the cross-link between methylglyoxal, dGuo, and a peptide (AcAVAGKAGAR) was also characterized. The mechanism of cross-link formation is likely to involve an Amadori rearrangement.

Figure 1

Protein and DNA adducts of methylglyoxal and glyoxal.

Figure 2

Structure of the dGuo, methylglyoxal, and N^α-AcLys cross-link 1.

Scheme 1. Synthesis of Cross-Link (1) Standards

Figure 3

UPLC chromatogram of the full scan mode (105 − 1100 Da), reconstructed ion chromatograms of SRM scan mode and MS³ fragment ions, and MS³ product ion spectrum of the dGuo-methylglyoxal-AcLys cross-link (1). (A) From the reaction of dGuo, AcLys, and methylglyoxal (1:1:4). (B) Authentic cross-link standards of 1.

Figure 4

(A) HPLC chromatogram for the reaction of dGuo, AcLys, and methylglyoxal in a 1:1:4 ratio (pH 7.4). (B) UPLC chromatogram of full scan mode (105 − 1100 Da), reconstructed UPLC-ESI -MS/MS and SRM (−116 Da) ion chromatograms from the reaction of dGuo, AcLys, and methylglyoxal (1:1:4). (C) HPLC chromatogram from the cross-linking reaction with a slow addition of methylglyoxal.

Figure 5

Proposed structures of the 2:1 adducts of methylglyoxal or glyoxal with dGuo.

Figure 6

(A) Full-scan spectrum from the reaction of dGuo, methylglyoxal, and the AcAVAGKAGAR peptide in 100 mM, pH 7.4 phosphate buffer. (B) Collision-induced dissociation (CID) mass spectrum of the m/z 524.3 [M + 2H]²⁺. This ion results from the in source, neutral loss of the deoxyribose (see Table 1 for theoretical b- and y-ion masses).

Figure 7

Products identified by the mass spectrometric analysis of the reaction of dGuo, methylglyoxal, and the AcAVAGKAGAR peptide in 100 mM, pH 7.4 phosphate buffer.

Scheme 2. Mechanism of CE-dGuo Formation

Figure 8

NMR analysis of the reaction of dGuo and methylglyoxal (A) and dGuo, AcLys, and methylglyoxal (B) in deuterated buffer (top) versus standards (bottom).

Scheme 3. Mechanism of Cross-Link (1) Formation

Figure 9

Model cross-links formed by methylglyoxal and related 1,2-dicarbonyls.