The Future of DNA Adductomic Analysis

Abstract

Covalent modification of DNA, resulting in the formation of DNA adducts, plays a central role in chemical carcinogenesis. Investigating these modifications is of fundamental importance in assessing the mutagenicity potential of specific exposures and understanding their mechanisms of action. Methods for assessing the covalent modification of DNA, which is one of the initiating steps for mutagenesis, include immunohistochemistry, ³²P-postlabeling, and mass spectrometry-based techniques. However, a tool to comprehensively characterize the covalent modification of DNA, screening for all DNA adducts and gaining information on their chemical structures, was lacking until the recent development of "DNA adductomics". Advances in the field of mass spectrometry have allowed for the development of this methodology. In this perspective, we discuss the current state of the field, highlight the latest developments, and consider the path forward for DNA adductomics to become a standard method to investigate covalent modification of DNA. We specifically advocate for the need to take full advantage of this new era of mass spectrometry to acquire the highest quality and most reliable data possible, as we believe this is the only way for DNA adductomics to gain its place next to the other "-omics" methodologies as a powerful bioanalytical tool.

Keywords: DNA adductomics; DNA adducts; DNA damage; chemical carcinogenesis; constant neutral loss; genotoxicity; high resolution accurate mass (HRAM) mass spectrometry.

Figure 1

Summary of a typical sample preparation and analysis workflow for DNA adductomics analysis.

Figure 2

(A) The dominant fragmentation pathway of nucleoside adducts is the neutral loss of the 2’-deoxyribose moiety; (B) and (C) Common fragmentation pathways of nucleobase adducts. (Base = nucleobase, A = modification, and dR = 2’-deoxyribose).

Figure 3

Examples of the type of DNA modifications that can be analyzed with mass spectrometry-based DNA adductomic approaches. The deoxyribose moiety of the structure is abbreviated as dR.

Figure 4

Illustration of different types of high resolution accurate mass (HRAM) DNA adductomic (MSⁿ) detection where N = 1 represents Full Scan, N = 2 represents Full Scan and MS/MS or MS/MS only, and N = 3 represents Full Scan, MS/MS, and MS/MS/MS. In this example, M is a nucleoside adduct with the general formula of M = X-G-dR where G is guanine, dR is the deoxyribose moiety, and X is the modification. In the Full Scan panel, the accurate mass of the DNA adduct ([M + H]⁺) can be extracted to generate a chromatogram and provide molecular formula information (this step is common to both DDA and DIA approaches). In the second panel, the MS/MS signal can either be extracted to generate a chromatogram (as in the case of a DIA approach) or provide MS/MS spectral data for the adduct (as in the case of a DDA approach). Finally, in the third panel, the MS/MS/MS fragmentation data can be used to indicate presence of a DNA adduct as well as provide structural confirmation/information (this is the final step for the DDA approach, while it is done in a separate injection in a DIA approach, focusing on candidate adducts identified in the first analysis).