Development of an untargeted DNA adductomics method by ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry


Cancer may be initiated by covalent modification of DNA by genotoxic molecules coming from diet, environment, inflammation, and other sources. For most of these genotoxicants there is little evidence of their identity. DNA adductomics is a new research field, aiming to screen for unknown DNA adducts by high resolution mass spectrometry (HRMS). However, due to the low abundance of DNA adducts, DNA adductomics presents several analytical challenges. In this work, a sensitive untargeted DNA adductomics method was developed by using ultra-high performance liquid chromatography (UHPLC) coupled via an electrospray ionization source (ESI) to a quadrupole-time of flight MS 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. A hybrid surface coated HSST3 premier column was tested for reducing adsorption of the DNA adducts. The optimized method was applied to analyse DNA adducts in calf thymus and cat colon DNA by performing a MSE acquisition and screening for loss of deoxyribose, both in-source and in the fragmentation spectra, and for the nucleobase fragment ions. The putative DNA adducts were matched with an in-house DNA adduct database. Thirteen DNA adducts were observed in DNA from calf thymus and cat colon, four of them never reported before, showing promise for the application of this untargeted method in future human studies.

Version notes

The paper has been updated by including an extra analysis on the comparison of an HSST3 column and an HSST3 premier column, the former showing the best performance. The paper has beeing also rephrased to give more focus on the potential of the method of identifying untargeted DNA adducts.


Supplementary material

Supplementary material
The supporting information contains several supplementary figures and tables: List of the tested chromatographic (Table S1) and mass spectrometric (Table S2) conditions; Settings for the pre-processing of the chromatographic runs with MZmine 2.53 (Table S3.1) and UNIFI (Table S3.2); Results of the chromatographic optimization in terms of signal intensity with HCOOH (Figure S4.1), CH3COOH (Figure S4.2), CH3COONH4 (Figure S4.3), HCOONH4 (Figure S4.4), NH4HCO3 (Figure S4.5); Results of the chromatographic optimization in terms of peak asymmetry factor (Figure S4.6), peak capacity (Figure S4.7), retention time standard deviation (Figure S4.8), resolution (Table S4.1); comparison of glass vs polyethylene bottles (Figure S4.9); Results of mass spectrometric optimization (Figure S5.1); Results of adsorption processes in terms of comparison among different low adsorption vials (Figure S6.1), comparison between the columns Premier HSST3 and HSST3 (Figure S6.2); Results of the identification of the DNA adducts in real samples (Table S7.1) and relative structures (Figure S7.2).