Derivatization with Girard reagent T combined with LC-MS/MS for the sensitive detection of 5-formyl-2'-deoxyuridine in cellular DNA - PubMed (original) (raw)

Derivatization with Girard reagent T combined with LC-MS/MS for the sensitive detection of 5-formyl-2'-deoxyuridine in cellular DNA

Haizheng Hong et al. Anal Chem. 2007.

Abstract

Nucleoside 5-formyl-2'-deoxyuridine (FodU) is a major thymidine lesion generated by reactive oxygen species. In vitro and in vivo replication studies revealed that FodU can be mutagenic. A reliable and sensitive quantification method is, therefore, important for assessing the biological implications of this lesion. However, the detection limit of FodU by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was relatively poor compared with those of other oxidative DNA base damages. In this paper we described a new approach for the highly sensitive detection of FodU. We derivatized FodU with Girard reagent T to form a hydrazone conjugate harboring a precharged quaternary ammonium moiety, which enabled the facile detection of the resulting conjugate by positive-ion electrospray ionization MS. We also showed that the combination of derivatization with LC-MS/MS on a linear-ion-trap mass spectrometer could allow for the quantification of FodU at a detection limit of 3-4 fmol, which is approximately 20-fold better than that for the direct analysis of the underivatized compound. By using isotope-labeled FodU as the internal standard and this derivatization method, we further quantified, by using LC-MS/MS, the yield of FodU formed in cellular DNA.

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Figures

Figure 1

Figure 1

The reaction of FodU with GirT.

Figure 2

Figure 2

(a) The HPLC trace for the separation of the reaction mixture where FodU was treated with GirT at a molar ratio of 1:2 at room temperature for 12 h. (b) ESI-MS of the ion of m/z 254 observed in MS/MS; shown in the inset are the MS/MS of the [M + H]+ ion of the hydrazone conjugate and the cleavage reactions for the formation of major fragment ions found in MS.

Figure 3

Figure 3

(a) Optimization of the molar ratios of GirT over FodU for the derivatization. GirT was mixed with FodU at molar ratios of 2:1, 10:1, 30:1, 100:1, 300:1, 1000:1 and 2000:1 in the presence of 10% acetic acid, and the reactions were continued at room temperature for 12 h. Plotted are the HPLC peak areas for FodU and the hydrazone conjugate. (b) Optimization of the derivatization time. GirT was mixed with FodU at a molar ratio of 10:1 in the presence of 10% acetic acid, and the reactions were carried out at room temperature for time periods of 10 min and 1, 2, 4, 12 and 24 h, respectively. Plotted are the HPLC peak areas for the hydrazone conjugate (the fractions were monitored by UV detection at 281 nm). The values represent the means ± SD from three independent measurements.

Figure 4

Figure 4

SICs for the monitoring of the m/z 370→254→195 [a, for unlabeled FodU-GirT hydrazone] and m/z 373→256→197 [b, for isotopically labeled FodU-GirT hydrazone] transitions of the digestion mixtures of the cellular DNA samples, which were extracted from Hela-S3 cells treated with 110 Gy of γ-rays. Shown in the insets are the MS of the ions of m/z 254 and 256 for the unlabeled and labeled hydrazones, respectively. The “*” in the structure shown in the inset of panel b designates that the nitrogen atoms are replaced with an “15N”.

Figure 5

Figure 5

Dose-dependent formation of FodU in Hela-S3 cells upon exposure to γ-rays. The values represent the means ± SD from three independent measurements.

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