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Characterization of the deoxyguanosine-lysine cross-link of methylglyoxal.

Petrova KV, Millsap AD, Stec DF, Rizzo CJ - Chem. Res. Toxicol. (2014)

Bottom Line: 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).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.

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Affiliation: Departments of Chemistry and Biochemistry, Center in Molecular Toxicology, and Vanderbilt-Ingram Cancer Center, Vanderbilt University , Nashville, Tennessee 37235, United States.

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.

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UPLC chromatogram ofthe 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-AcLyscross-link (1). (A) From the reaction of dGuo, AcLys,and methylglyoxal (1:1:4). (B) Authentic cross-link standards of 1.
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fig3: UPLC chromatogram ofthe 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-AcLyscross-link (1). (A) From the reaction of dGuo, AcLys,and methylglyoxal (1:1:4). (B) Authentic cross-link standards of 1.

Mentions: The mass spectrum of cross-link 1 (m/z 508.2 [M – H]1−) andits fragmentation in negative ion mode is shown in Figure 3B. The initial characteristic loss of the deoxyriboseunit (−116 Da, 508 → 392) is followed by decarboxylationof the lysine portion (−44, 392 → 349). Interestingly,the amide bond involving the lysine side chain readily fragmented(−145, 349 → 204) at low collision energy (10 eV). Themass spectra of both diastereomers were identical. An authentic standardof the CE-dGuo adduct was also synthesized from the reaction of dGuoand glyceraldehyde.41 The mass spectral fragmentation of CE-dGuo in negative ion modeinvolved decarboxylation (−44, 338 → 294) followed byneutral loss of the deoxyribose (−116, 294 → 178) (seeFigure S17 of the Supporting Information), which is consistent with a previous analysis.7,8 Cross-link 1 and CE-dGuo adducts were also characterized by 1D- and 2D-1H NMR spectroscopy (see Figure S8–S13 of the Supporting Information).


Characterization of the deoxyguanosine-lysine cross-link of methylglyoxal.

Petrova KV, Millsap AD, Stec DF, Rizzo CJ - Chem. Res. Toxicol. (2014)

UPLC chromatogram ofthe 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-AcLyscross-link (1). (A) From the reaction of dGuo, AcLys,and methylglyoxal (1:1:4). (B) Authentic cross-link standards of 1.
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fig3: UPLC chromatogram ofthe 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-AcLyscross-link (1). (A) From the reaction of dGuo, AcLys,and methylglyoxal (1:1:4). (B) Authentic cross-link standards of 1.
Mentions: The mass spectrum of cross-link 1 (m/z 508.2 [M – H]1−) andits fragmentation in negative ion mode is shown in Figure 3B. The initial characteristic loss of the deoxyriboseunit (−116 Da, 508 → 392) is followed by decarboxylationof the lysine portion (−44, 392 → 349). Interestingly,the amide bond involving the lysine side chain readily fragmented(−145, 349 → 204) at low collision energy (10 eV). Themass spectra of both diastereomers were identical. An authentic standardof the CE-dGuo adduct was also synthesized from the reaction of dGuoand glyceraldehyde.41 The mass spectral fragmentation of CE-dGuo in negative ion modeinvolved decarboxylation (−44, 338 → 294) followed byneutral loss of the deoxyribose (−116, 294 → 178) (seeFigure S17 of the Supporting Information), which is consistent with a previous analysis.7,8 Cross-link 1 and CE-dGuo adducts were also characterized by 1D- and 2D-1H NMR spectroscopy (see Figure S8–S13 of the Supporting Information).

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Departments of Chemistry and Biochemistry, Center in Molecular Toxicology, and Vanderbilt-Ingram Cancer Center, Vanderbilt University , Nashville, Tennessee 37235, United States.

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.

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