# Identifying Novel DNA Adducts in Amphipods and Developing Sample Preparation for Adductomics Using Dispersive Solid-Phase Extraction

## TL;DR

This paper introduces a new method for identifying DNA adducts in amphipods and improves sample preparation for DNA adductomics using dispersive solid-phase extraction.

## Contribution

The study introduces a novel DNA adduct identification workflow and proposes dispersive solid-phase extraction for DNA adductomics in nonmammalian species.

## Key findings

- 16 DNA adducts were identified, including 10 novel modifications in amphipod DNA.
- Dispersive solid-phase extraction reduced matrix interferences and improved LC–HRMS signal response by up to 170%.
- The method is a simple alternative to conventional cleanup techniques for DNA adduct analysis in crustaceans.

## Abstract

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.