Identifying Novel DNA Adducts in Amphipods and Developing Sample Preparation for Adductomics Using Dispersive...

The growing use of environmental DNA adductomics for exposure assessment emphasizes the need for improved methods that are adapted to chemically and biologically diverse samples. While liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) has advanced DNA adductome analysis, sample cleanup methods remain challenging, especially in nonmammalian models. Here, we used amphipod Monoporiea affinis, a sentinel species in biological effect studies, as a surrogate crustacean species for the method development. It is particularly suitable because its chitinous tissues and high lipid content present a challenging matrix for DNA extraction. We addressed the following objectives: (i) to propose adduct structures by combining multiple tools for DNA adduct screening and (ii) to evaluate the applicability of dispersive solid-phase extraction (d-SPE) as a cleanup step for DNA adductome analysis. Toward the first objective, we integrated open-source software nLossFinder with a recently introduced DNA adductomics database (GitLab) to enhance the structural identification of unknown adducts. A combination of accurate mass data and MS2-fragmentation allowed differentiation of the complex mixture of nucleoside adducts, facilitating the structural identification of 16 DNA adducts, including 10 modifications on amphipod DNA reported for the first time. Toward the second objective, we introduced d-SPE as a novel cleanup approach for DNA adduct analysis in whole-body crustacean samples. Using Z-sep+ as the d-SPE sorbent, we demonstrated a major reduction of matrix interferences, including phospholipids, and enhanced sensitivity toward DNA adducts detection, leading to improved LC-HRMS signal response by up to 170%. d-SPE offers a simple alternative to conventional methods like standard SPE and liquid-liquid extraction, making it a valuable tool for DNA adductomics in environmental monitoring and aiding high-throughput capacity, especially while handling large numbers of samples. Further studies should include validation of this method for other species and DNA modifications. These advancements underscore the potential of the proposed data analysis workflow and d-SPE for improving mass spectrometry-based DNA adductomics in environmental monitoring, paving the way for more accurate and comprehensive exposure assessments across diverse species and environmental conditions.