Development of a DNA Adductome Mass Spectral Database

Abstract

Mass spectrometry-based DNA adductomics is an emerging approach for the human biomonitoring of hazardous chemicals. A mass spectral database of DNA adducts will be created for the scientific community to investigate the associations between chemical exposures, DNA damage, and disease risk.

Graphical Abstract

Environmental and dietary genotoxicants, endogenous electrophiles, together with ionizing and non-ionizing radiation, continuously damage the genome. Fortunately, cells have DNA repair systems to cope with the many different types of DNA lesions. However, DNA damage in cancer-related genes that escapes repair can induce mutations during cell division and initiate the carcinogenic process.^1,2 The identification of DNA adducts and their potential to induce mutations are among the first steps in the risk assessment of harmful chemicals. DNA adducts are dosimeters of genotoxicants and are used as vital biomarkers in the formulation of public health policy designed to decrease chemical exposures and cancer incidence. DNA adducts also serve as candidate biomarkers to associate polymorphisms in genes that encode enzymes involved in the metabolism of carcinogens and DNA repair with individual susceptibilities of cancer risk. In addition, DNA adducts serve as biomarkers to assess the efficacy of chemoprevention protocols and chemotherapeutic agents in precision medicine.

DNA adducts of aflatoxin B₁ (AFB₁) and aristolochic acid I (AA-I) and their characteristic mutational signatures have been used to firmly establish their causative roles in liver and renal cancer, respectively, in Asia, where the exposure levels are high.^1,2 In contrast, populations in the United States and other western countries are exposed to lower levels of a wide array of potential cancer-causing agents. Targeted mass spectrometry methods conducted by investigators worldwide have successfully measured different types of DNA adducts formed from a wide range of chemicals present in the environment, tobacco smoke (e.g., 4-aminobiphenyl, 4-ABP), well-done cooked meats (e.g., 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, PhIP), or endogenously produced electrophiles (e.g., malondialdehyde, MDA).³ These analyses usually target one, or up to several adducts per assay, but fail to capture the totality of adducts in the genome. Adopting an untargeted approach can facilitate the identification of expected and unexpected DNA adducts generated from exposures, which may be risk factors for diseases, including cancer. With the advancement of high-resolution mass spectrometry (HRMS) instrumentation, the untargeted scanning of DNA damage, termed “DNA adductomics”, has the potential to simultaneously screen for DNA adducts of exogenous genotoxicants, endogenous electrophiles, and chemotherapeutic drugs in a single assay.⁴

The emerging DNA adductomic technique is a promising technology but hindered by the lack of a publicly available mass spectral database for characterization and identificationof DNA adducts. Open-source MS-based metabolomics databases, such as MassBank of North America (MoNA) (http://mona.fiehnlab.ucdavis.edu/), Human Metabolome Database (HMDB) (http://www.hmdb.ca/), and proprietary small molecule libraries from the National Institute of Standards and Technology (NIST) (https://chemdata.nist.gov/) have been used for many years to analyze and process metabolomic data. Our goal is to curate a comprehensive database of DNA adducts and their mass spectra, which will be freely available to the public to facilitate data-mining for the identification of DNA adducts.

The database will be comprised of 2′-deoxyribonucleoside and nucleobase adducts formed from environmental and dietary genotoxicants, and endogenous electrophiles, but will also include adducts of several chemotherapeutic drugs and their DNA crosslinks, together with RNA adduct standards. HR MSⁿ spectra of authentic DNA adduct standards will be acquired on Orbitrap and Q-TOF instruments, which are the two most commonly used HRMS platforms for DNA adduct analyses. High quality curated and annotated spectra will be submitted to the MoNA open-source spectral library, and spectra acquired by Orbitrap instrumentation will also be integrated into the open-source mzCloud (Thermo Fisher Scientific) database. Each spectral library entry will include the common and IUPAC names, canonical and isomeric Simplified Molecular-Input Line-Entry System (SMILES) notations, IUPAC International Chemical Identifier (InChI), names and affiliations of the contributors, and MS scanning parameters (TOC figure). The relevant metadata will also be uploaded to the PubChem database, from where the metadata and spectral information will be made available in HMDB.

The workflow strategy⁴ is illustrated in the TOC Figure, whereby Orbitrap and Q-TOF MS spectra of high purity and fully characterized synthetic standards are acquired at the MS, MS², and MS³ (Orbitrap only) levels using loop injections. Orbitrap MS² spectra of collision-induced dissociation (CID) and high-energy CID (HCD) fragmentations are collected with collision energies (CE) ranging from 0 – 50% and 0 – 100%, respectively. MS³ spectra, collected on the 3 most intense MS² ions above a 50% threshold, are collected at 30 and 60% MS² HCD using 20 – 80 % MS³ HCD, and 30% MS² CID using 15 – 45% MS³ CID. Q-TOF MS² spectra is collected at CE of 0 – 40 V. High-intensity MSⁿ scans are automatically selected for data processing using a custom informatics workflow, including the use of MS-Finder software (http://prime.psc.riken.jp/Metabolomics_Software/MS-FINDER/index2.html) to assign molecular formulae to the fragment ions. After annotations are performed, consensus spectra at all CE values using the theoretical m/z and average intensities will be uploaded to the open-source spectral libraries. The identity of the contributed synthetic standards will be checked by measurement of the accurate mass of the protonated compound.

Thirty-six collaborators worldwide have generously agreed to provide DNA standards to populate the DNA adductome database. We are actively seeking investigators to contribute additional reference standards, and individuals interested in participating should contact the corresponding authors. A communication on the DNA adduct database will be published and list all contributing investigators, the DNA adducts they have provided, and with each investigators’ approval, their contact information to facilitate the dissemination of reference standards. Our workflow and spectral library building code will be provided to assist investigators in their efforts to incorporate spectra and metadata of newly identified and characterized DNA adducts into MoNA and PubChem.

The validated DNA adduct mass spectral library will serve as a powerful tool allowing investigators to characterize DNA adductome profiles, and assist in identification of unknown putative DNA adducts through the implementation of data analysis tools, such as fragmentation trees and molecular network analysis.⁵ In vivo DNA adduct levels are typically very low, making untargeted detection challenging, however, improved methodologies are under development to make these measurements possible.⁴ The database will provide a critical resource for DNA adduct identification and profiling capability necessary to improve our understanding of the relationships between external and internal chemical exposures and disease risk.