Journal
ANALYTICAL CHEMISTRY
Volume 79, Issue 1, Pages 322-326Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac061465w
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Funding
- NATIONAL CANCER INSTITUTE [R01CA096906] Funding Source: NIH RePORTER
- NCI NIH HHS [R01 CA096906, R01 CA96906, R01 CA096906-04] Funding Source: Medline
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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 similar to 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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