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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.

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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(A) HPLC chromatogram for the reaction of dGuo, AcLys, and methylglyoxalin 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 (−116Da) ion chromatograms from the reaction of dGuo, AcLys, and methylglyoxal(1:1:4). (C) HPLC chromatogram from the cross-linking reaction witha slow addition of methylglyoxal.
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fig4: (A) HPLC chromatogram for the reaction of dGuo, AcLys, and methylglyoxalin 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 (−116Da) ion chromatograms from the reaction of dGuo, AcLys, and methylglyoxal(1:1:4). (C) HPLC chromatogram from the cross-linking reaction witha slow addition of methylglyoxal.

Mentions: Thecross-linking reaction was initially investigated using a 4-fold molarexcess of methylglyoxal in phosphate buffer (100 mM, pH 7.4) at 37°C for 7 days. HPLC analysis of the reaction showed three predominantproducts (Figure 4A) having retention times(tr) between ∼16 and 19 min. UPLC-MSanalysis (Figure 4B) of the reaction revealedthat the major products had masses consistent with 2:1 methylglyoxal-dGuoadducts (m/z 410 [M – H]1–). Minor products, which coeluted with the 2:1 adducts,had masses consistent with a 1:1 adduct between methylglyoxal and dGuo (m/z 338 [M – H]1–) and are likely to be the diastereomeric 1,N2-cyclic adducts (MG-dGuo) and CE-dGuo. Under the conditionsof the reaction, the cross-links were not detectable by HPLC (Figure 4A, insert) but could be readily identified by UPLC-ESI-MSn (Figure 4B). Withstandards in hand, we identified the diastereomeric cross-links ofinterest (Figure 3A), and their mass spectrawere identical to those of the standard (Figure 3B).


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

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

(A) HPLC chromatogram for the reaction of dGuo, AcLys, and methylglyoxalin 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 (−116Da) ion chromatograms from the reaction of dGuo, AcLys, and methylglyoxal(1:1:4). (C) HPLC chromatogram from the cross-linking reaction witha slow addition of methylglyoxal.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4060920&req=5

fig4: (A) HPLC chromatogram for the reaction of dGuo, AcLys, and methylglyoxalin 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 (−116Da) ion chromatograms from the reaction of dGuo, AcLys, and methylglyoxal(1:1:4). (C) HPLC chromatogram from the cross-linking reaction witha slow addition of methylglyoxal.
Mentions: Thecross-linking reaction was initially investigated using a 4-fold molarexcess of methylglyoxal in phosphate buffer (100 mM, pH 7.4) at 37°C for 7 days. HPLC analysis of the reaction showed three predominantproducts (Figure 4A) having retention times(tr) between ∼16 and 19 min. UPLC-MSanalysis (Figure 4B) of the reaction revealedthat the major products had masses consistent with 2:1 methylglyoxal-dGuoadducts (m/z 410 [M – H]1–). Minor products, which coeluted with the 2:1 adducts,had masses consistent with a 1:1 adduct between methylglyoxal and dGuo (m/z 338 [M – H]1–) and are likely to be the diastereomeric 1,N2-cyclic adducts (MG-dGuo) and CE-dGuo. Under the conditionsof the reaction, the cross-links were not detectable by HPLC (Figure 4A, insert) but could be readily identified by UPLC-ESI-MSn (Figure 4B). Withstandards in hand, we identified the diastereomeric cross-links ofinterest (Figure 3A), and their mass spectrawere identical to those of the standard (Figure 3B).

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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