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Original Research ARTICLE Provisionally accepted The full-text will be published soon. Notify me

Front. Chem. | doi: 10.3389/fchem.2019.00658

Targeted High Resolution LC/MS3 Adductomics Method for the Characterization of Endogenous DNA Damage

 Andrea Carra'1,  Valeria Guidolin1,  Romel P. Dator1, Pramod Upadhyaya1, Fekadu Kassie1,  Peter W. Villalta1* and Silvia Balbo1*
  • 1Masonic Cancer Center, University of Minnesota, United States

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 all 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 all known endogenous adducts, and neutral loss MS3 triggering to profile all DNA modifications. In this method, detection of endogenous DNA adducts is performed by observation of their corresponding MS3 neutral loss triggered events and the relative quantitation using the corresponding full scan extracted ion chromatograms. The method’s inclusion list consists of all known endogenous DNA adducts, compiled and reported here, as well as adducts specific to tobacco exposure 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) and its effects are enhanced by induction of inflammation from co-exposure of the animals 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 adducts due to NNK exposure. The detected DNA adducts were then targeted for PRM-MS2 analysis and precise relative quantitation was measured, demonstrating a general workflow for analysis of endogenous DNA adducts.

Keywords: Mass Spectrometry, DNA adductomics, Cancer, Lipid Peroxidation, Inflammation

Received: 03 Jul 2019; Accepted: 17 Sep 2019.

Copyright: © 2019 Carra', Guidolin, Dator, Upadhyaya, Kassie, Villalta and Balbo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence:
Dr. Peter W. Villalta, Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, Minnesota, United States, villa001@umn.edu
Prof. Silvia Balbo, Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, Minnesota, United States, balbo006@umn.edu