Quantitation of DNA Adducts by Stable Isotope Dilution Mass Spectrometry

Tretyakova, Natalia; Goggin, Melissa; Sangaraju, Dewakar; Janis, Gregory C.

doi:10.1021/tx3002548

Cited by 107 publications

(131 citation statements)

References 167 publications

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“…46 A calibration curve was developed by plotting the ratio of the cross-link ( S )- 1 (0.25 – 100 ng)/[ 13 C 3 15 N 1 ]-( S )- 1 (50 ng) versus the integrated ratio of ( S )- 1 ( m / z 508 → 392)/[ 13 C 3 15 N 1 ]-( S )- 1 ( m / z 512 → 396). The calibration curve was linear over the concentration range examined ( r 2 = 0.998, Figure S18 and S19 of the Supporting Information).…”

Section: Resultsmentioning

confidence: 99%

Characterization of the Deoxyguanosine–Lysine Cross-Link of Methylglyoxal

Petrova

Millsap

Stec

et al. 2014

Chem. Res. Toxicol.

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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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Section: Resultsmentioning

confidence: 99%

Characterization of the Deoxyguanosine–Lysine Cross-Link of Methylglyoxal

Petrova

Millsap

Stec

et al. 2014

Chem. Res. Toxicol.

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“…LC-MS/MS coupled with the isotope-dilution technique constitutes the most reliable analytical method for the quantification of modified nucleosides in cellular and tissue DNA [31]. Although synthetic methods were previously developed for the synthesis of dJ and for its incorporation into ODNs [32-34], the methods involve a multi-step synthesis and are not readily amenable for the preparation of stable isotope-labeled dJ.…”

Section: Resultsmentioning

confidence: 99%

Quantitative Mass Spectrometry-Based Analysis of β-D-Glucosyl-5-Hydroxymethyluracil in Genomic DNA of Trypanosoma brucei

Liu

Cliffe

et al. 2014

J. Am. Soc. Mass Spectrom.

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β-D-glucosyl-5-hydroxymethyluracil (base J) is a hyper-modified nucleobase found in the nuclear DNA of kinetoplastid parasites. With replacement of a fraction of thymine in DNA, J is localized primarily in telomeric regions of all organisms carrying this modified base. The biosynthesis of J occurs in two putative steps: First, a specific thymine in DNA is recognized and converted into 5-hydroxymethyluracil (5-HmU) by J-binding proteins (JBP1 and JBP2); a glucosyl transferase (GT) subsequently glucosylates the 5-HmU to yield J. Although several recent studies revealed the roles of internal J in regulating transcription in kinetoplastids, functions of telomeric J and proteins involved in J synthesis remain elusive. Assessing the functions of base J and understanding fully its biosynthesis necessitate the measurement of its level in cells and organisms. In this study, we reported a reversed-phase HPLC coupled with tandem mass spectrometry (LC-MS/MS) method, together with the use of a surrogate internal standard (β-D-glucosyl-5-hydroxymethyl-2′-deoxycytidine, 5-gHmdC), for the accurate detection of β-D-glucosyl-5-hydroxymethyl-2′-deoxyuridine (dJ) in Trypanosoma brucei DNA. For comparison, we also measured the level of the precursor for dJ synthesis, i.e. 5-hydroxymethyl-2′-deoxyuridine (5-HmdU). We found that base J was not detectable in the JBP-null cells while it replaced approximately 0.5% thymine in wild-type cells, which was accompanied with a markedly decreased level of 5-HmdU in JBP1/JBP2-null strain relative to the wild-type strain. These results provided direct evidence supporting that JBP proteins play an important role in oxidizing thymidine to form 5-HmdU, which facilitated the generation of dJ. This is the first report about the application of LC-MS/MS for the quantification of base J. The analytical method built a solid foundation for dissecting the molecular mechanisms of J biosynthesis and assessing the biological functions of base J in the future.

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“…16,19–21 We and other laboratories have developed targeted LC-MS methods to qualitatively and quantitatively analyze DNA adducts for a number of human carcinogens. 20,22,23 However, to broadly screen for DNA adducts of multiple classes of carcinogens, an “adductomics” approach is required where robust and universal methods of sample preparation and unbiased MS scanning features can be employed to characterize an array of DNA adducts. The recent improvements in MS scanning acquisition capabilities and instrument sensitivity have set the stage for the use of LC-MS in high-throughput applications in DNA adductomics.…”

Section: Introductionmentioning

confidence: 99%

Data-Independent Mass Spectrometry Approach for Screening and Identification of DNA Adducts

2017

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Long-term exposures to environmental toxicants and endogenous electrophiles are causative factors for human diseases including cancer. DNA adducts reflect the internal exposure to genotoxicants and can serve as biomarkers for risk assessment. Liquid chromatography-multistage mass spectrometry (LC-MSn) is the most common method for biomonitoring DNA adducts, generally targeting single exposures and measuring up to several adducts. However, the data often provide limited evidence for a role of a chemical in the etiology of cancer. An “untargeted” method is required that captures global exposures to chemicals, by simultaneously detecting their DNA adducts in the genome; some of which may induce cancer-causing mutations. We established a wide selected ion monitoring tandem mass spectrometry (Wide-SIM/MS2) screening method utilizing ultra-performance-LC nanoelectrospray ionization Orbitrap MSn with on-line trapping to enrich bulky, non-polar adducts. Wide-SIM scan events are followed by MS2 scans to screen for modified nucleosides by co-eluting peaks containing precursor and fragment ions differing by -116.0473 Da, attributed to the neutral loss of deoxyribose. Wide-SIM/MS2 was shown to be superior in sensitivity, specificity, and breadth of adduct coverage to other tested adductomic methods with detection possible at adduct levels as low as 4 per 109 nucleotides. Wide-SIM/MS2 data can be analyzed in a “targeted” fashion by generation of extracted ion chromatograms or in an “untargeted” fashion where a chromatographic peak-picking algorithm can be used to detect putative DNA adducts. Wide-SIM/MS2 successfully detected DNA adducts, derived from chemicals in the diet and traditional medicines and from lipid peroxidation products, in human prostate and renal specimens.

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Quantitation of DNA Adducts by Stable Isotope Dilution Mass Spectrometry

Cited by 107 publications

References 167 publications

Characterization of the Deoxyguanosine–Lysine Cross-Link of Methylglyoxal

Characterization of the Deoxyguanosine–Lysine Cross-Link of Methylglyoxal

Quantitative Mass Spectrometry-Based Analysis of β-D-Glucosyl-5-Hydroxymethyluracil in Genomic DNA of Trypanosoma brucei

Data-Independent Mass Spectrometry Approach for Screening and Identification of DNA Adducts

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