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Abstract
Background: Genotoxicants originating from inflammation, diet, and environment can covalently modify DNA, possibly initiating the process of carcinogenesis. DNA adducts have been known for long, but the old methods allowed to target only a few known DNA adducts at a time, not providing a global picture of the “DNA adductome”. DNA adductomics is a new research field, aiming to screen for unknown DNA adducts by high resolution mass spectrometry (HRMS). However, DNA adductomics presents several analytical challenges such as the need for high sensitivity and for the development of effective screening approaches to identify novel DNA adducts.
Results: In this work, a sensitive untargeted DNA adductomics method was developed by using ultra-high performance liquid chromatography (UHPLC) coupled via an ESI source to a quadrupole-time of flight mass spectrometric instrumentation. Mobile phases with ammonium bicarbonate gave the best signal enhancement. The MS capillary voltage, cone voltage, and detector voltage had most effect on the response of the DNA adducts. A low adsorption vial was selected for reducing analyte loss. Hybrid surface-coated analytical columns were tested for reducing adsorption of the DNA adducts. The optimized method was applied to analyse DNA adducts in calf thymus, cat colon, and human colon DNA by performing a MSE acquisition (all-ion fragmentation acquisition) and screening for the loss of deoxyribose and the nucleobase fragment ions. Fifty-four DNA adducts were tentatively identified, hereof 38 never reported before.
Significance: This is the first untargeted DNA adductomics study on human colon tissue, and one of the few untargeted DNA adductomics studies in the literature reporting the identification of such a high number of unknowns. This demonstrates promising results for the application of this sensitive method in future human studies for investigating novel potential cancer-causing factors.
Supplementary materials
Title
Supplementary material
Description
List of the tested chromatographic (Table S1) and mass spectrometric (Table S2) conditions; Settings for the pre-processing with MZmine 2.53 (Table S3) and UNIFI 1.9.4.053 (Table S4); Results of the chromatographic optimization in terms of signal intensity with HCOOH (Figure S1), CH3COOH (Figure S2), CH3COONH4 (Figure S3), HCOONH4 (Figure S4), NH4HCO3 (Figure S5); Results of the chromatographic optimization in terms of peak asymmetry factor (Figure S6), peak capacity (Figure S7), retention time standard deviation (Figure S8), resolution (Table S5); comparison of glass vs polyethylene bottles (Figure S9); Results of mass spectrometric optimization (Figure S10); Results of adsorption processes in terms of comparison among different low adsorption vials (Figure S11-S12), comparison between the columns Premier HSS T3, HSS T3, Premier BEH and BEH (Figure S13-S14); Instrumental LOD of the DNA adduct reference standards (Table S6); Results of the identification of the DNA adducts in real samples (Table S7).
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