- Liquid chromatography HRMS analysis and data processing, including low- and high-mass adduct detection \cite{Martella2023}, quantification and identification using open-source databases \cite{La2022,Guo_2020}. Typically, reversed phase chromatography coupled to an Orbitrap HRMS instrumentation can be used for this purpose, employing data-dependent acquisition with an inclusion list or data-independent acquisition. The resulting high-resolution accurate mass (HRAM) data allows for increased selectivity due to the ability to differentiate the adduct ion signals from isobaric background ions. The accurate mass measurements further provide sufficient information to determine the molecular formula of the adduct analytes, which are useful in the structural characterization of the adducts. Open-source software for peak screening of the DNA adducts is available \cite{Sousa2021} for non-targeted detection of DNA adducts based on characteristic neutral loss (deoxyribose moiety, 116.0474 Da) and without any prior knowledge of the adducts. To reduce false positives, data filtering is performed using retention time alignment, accurate mass window, MS/MS fragments and any associated metadata available. The output data are normalized peak areas for specific adducts that can be used for the downstream statistical evaluations (Figure 2). The data format is typically a matrix, with adduct relative abundance and samples (i.e., individuals) given in columns and rows or vice-versa.
- Statistical analysis for the DNA adductome of each sentinel species used for monitoring purposes should include the following steps: (1) screening the output data to exclude non-varying adducts; (2) identification of the background variability; and (3) evaluation of the test samples against the background variability (i.e., reference state). However, currently, there are no established methods for deriving environmental quality standards from the omics data \cite{Henke2023,Machuca-SepĂșlveda2023,Ebner2021}; therefore, different approaches should be evaluated and compared using data for different species and environmental settings.
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