Masshunter workstation data software

Extraction of CE₂ (4-OHE₂ and 2-OHE₂) was performed according to the previous study with minor revision. In brief, approximately 1 g of ground mammary tissue was suspended in 2 ml of 50 mM ammonium acetate (pH 5.0) followed by addition of methanol with the final concentration of 60%. The mixture was extracted with 4 ml hexane to remove lipids. The aqueous phase was then diluted to an approximate final concentration of 25 % methanol with 30 mM ammonium acetate buffer, pH 4.4, containing 2 mg/ml ascorbic acid (to minimized oxidation of CE). The resulting solution was filtered through a 10 kDa MWCO ultrafilter to reduced protein interference and turbidity. Sample was immediately run on HPLC and the Q-TOF mass spectrometry with the following parameter: autosampler temperature, 4 °C; injection volume: 2 µl; ESI mode and voltage: 4000 V (−) ion mode; pump solvent: 50% methanol and 50% acetonitrile; flow rate: 0.5 ml/min. Data was acquired at a rate of 2.5 spectra/s with a stored mass range of m/z 50–1500. Data was collected and analyzed using Agilent MassHunter Workstation Data software.

Extraction of depurinating DNA adducts was performed according to the previous study with minor revision. Briefly, approximately 1 g of ground mammary tissue was suspended in 3 ml of 1 % acetic acid and incubated 5 hours at room temperature. Afterwards, the depurinating DNA adducts, 4-OHE₂-1-N3Ade and 4-OHE₂-1-N7Gua, were Soxhlet extracted with methanol/chloroform (1:1) for 24 hours. The 4-OHE₂-1-N3Ade and 4-OHE₂-1-N7Gua were then separated by HPLC according to the references of 4-OHE₂-1-N3Ade and 4-OHE₂-1-N7Gua. The purified adducts were subjected to a Q-TOF mass spectrometry with following parameter: autosampler temperature, 4 °C; injection volume: 4 µl; ESI mode and voltage: 4000 V (+) ion mode; pump solvent: 50% methanol and 50% acetonitrile; flow rate: 0.5 ml/min. Data was acquired at a rate of 2.5 spectra/s with a stored mass range of m/z 50–1500. Data was collected and analyzed using Agilent MassHunter Workstation Data software. The peak values of 4-OHE₂-1-N3Ade and 4-OHE₂-1-N7Gua showed the relative abundance of these adducts derived from different samples. Unmodified adenine or guanine was used as the loading control.