DNA can be damaged through covalent modifications of the nucleobases by endogenous processes. These modifications, commonly referred to as DNA adducts, can persist and may lead to mutations, and ultimately to the initiation of cancer. A screening methodology for the majority of known endogenous DNA adducts would be a powerful tool for investigating the etiology of cancer and for the identification of individuals at high-risk to the detrimental effects of DNA damage. This idea led to the development of a DNA adductomic approach using high resolution data-dependent scanning, an extensive MS2 fragmentation inclusion list of known endogenous adducts, and neutral loss MS3 triggering to profile all DNA modifications. In this method, the detection of endogenous DNA adducts is performed by observation of their corresponding MS3 neutral loss triggered events and their relative quantitation using the corresponding full scan extracted ion chromatograms. The method's inclusion list consists of the majority of known endogenous DNA adducts, compiled, and reported here, as well as adducts specific to tobacco exposure included to compare the performance of the method with previously developed targeted approaches. The sensitivity of the method was maximized by reduction of extraneous background signal through the purification and minimization of the amount of commercially obtained enzymes used for the DNA hydrolysis. In addition, post-hydrolysis sample purification was performed using off-line HPLC fraction collection to eliminate the highly abundant unmodified bases, and to avoid introduction of plasticizers found in solid-phase extraction cartridges. Also, several instrument parameters were evaluated to optimize the ion signal intensities and fragmentation spectra quality. The method was tested on an animal model of lung carcinogenesis where A/J mice were exposed to the tobacco specific lung carcinogen 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK) with its effects enhanced by co-exposure to the pro-inflammatory agent lipopolysaccharide (LPS). Lung DNA were screened for endogenous DNA adducts known to result from oxidative stress and LPS-induced lipid peroxidation, as well as for adducts due to NNK exposure. The relative quantitation of the detected DNA adducts was performed using parallel reaction monitoring MS2 analysis, demonstrating a general workflow for analysis of endogenous DNA adducts.
Bibliographical noteFunding Information:
We thank Dr. Laura Maertens and Andrew Casey Floeder for their contribution in the DNA sample extraction and preparation for DNA adductomics. We thank FK Lab Members for their contribution in the animal experiments. We thank Dr. Fung-Lung Chung for donating the 6-(1-Hydroxyhexanyl)-8-hydroxy-1, N 2-propano-2-deoxyguanosine standard. We thank Robert Carlson for assistance with the generation of figure graphics. Funding. This work was supported by University of Minnesota start-up funds for SB and salary support for PV was provided by the U.S. National Institutes of Health and National Cancer Institute [Grant R50-CA211256]. Mass spectrometry was carried out in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center, supported in part by the U.S. National Institutes of Health and National Cancer Institute [Cancer Center Support Grant CA-77598].
This work was supported by University of Minnesota start-up funds for SB and salary support for PV was provided by the U.S. National Institutes of Health and National Cancer Institute [Grant R50-CA211256]. Mass spectrometry was carried out in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center, supported in part by the U.S. National Institutes of Health and National Cancer Institute [Cancer Center Support Grant CA-77598].
© Copyright © 2019 Carrà, Guidolin, Dator, Upadhyaya, Kassie, Villalta and Balbo.
- DNA adductomics
- DNA damage
- lipid peroxidation
- mass spectrometry
- tobacco carcinogens