LC-MS/MS-Based Adductomic Approaches for Assessing DNA Damage
- Author(s): Cui, Yuxiang;
- Advisor(s): Wang, Yinsheng;
- et al.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been widely utilized in the analysis of DNA adducts. With recent developments in mass spectrometry instrumentation, DNA adductomics has emerged as an active field of research. To date, the majority of DNA adductomic methods involved the use of untargeted approaches for the discovery and screening of DNA adducts. Meanwhile, the demands for sensitive and quantitative measurements of DNA adducts persist. As more adducts become identified and characterized, targeted DNA adductomic approaches can become more comprehensive so as to address such demands.
In Chapter 2, I studied the collision-induced dissociations of regioisomeric DNA alkylation adducts in their nucleoside form with alkyl groups of various sizes. Depending on the site of alkylation and the size of the alkyl group, some regioisomeric adducts can be differentiated by MS/MS/MS, whereas many of them remain undistinguishable. Therefore, liquid chromatography constitutes an important basis for the separation and differentiation of regioisomeric DNA adducts.
In Chapter 3, the regioisomeric alkylation adducts along with other DNA adducts were incorporated into a scheduled selected-reaction monitoring (SRM)-based LC-MS/MS method for targeted analysis of the DNA adductome. Using canonical nucleosides as references, I established the iRT scores for alkylated DNA adducts, which is the basis for retention time prediction. The scheduled SRM-based method can allow for robust quantification of more adducts in one LC-MS/MS run.
In order to achieve reliable quantification, sample preparation is also a crucial element in DNA adduct analysis. In Chapters 4 and 5, the sample preparation workflows for a few DNA lesions that are prone to artifactual generation, 8,5′-cyclopurine-2′-deoxynucleosides (cPus) and 2′-deoxyinosine, were examined. I addressed possible sources of artifacts, highlighted the importance of offline HPLC enrichment, and evaluated the enzymatic release efficiencies of the lesions from DNA. For cPus, the release is the major challenge for reliable quantification. A calibration curve with lesion-containing ODN should be established in parallel with the analyses of DNA samples of interest. For dI, quantitative release can be achieved, although caution should be exerted to reduce background contamination.