Jet stream

For analyte detection a Triple Quadrupole LC/MS G6460A, equipped with an electrospray ion (ESI) source with JetStream (Agilent Technologies, Santa Clara, USA), was used. The mode of detection was a dynamic multiple reaction monitoring (dMRM) with a cycle time of 750 ms in a positive and negative ionization mode.
The drying gas temperature and flow of the ESI source were set at 250°C and 12 L/min respectively, sheath gas temperature and flow were 400°C and 12 L/min. The nebulizer was operated at a pressure of 50 psi and the nozzle voltage was set to 500 V in the positive mode and 0 V in negative mode. The capillary voltage was 4,000 V in the positive mode and 2,500 V in the negative mode. The electron multiplier voltage was set to 400 V (positive) and 500 V (negative). For the fragmentation of precursor ions in the collision cell, nitrogen was used as collision gas. The most abundant precursor ions were [M+H]⁺ and [M−H]⁻ in positive and negative ionization mode, respectively, for all analytes. Fragmentor voltage, collision energy, and cell accelerator voltage were optimized individually for each compound or transition by manually injecting standard substances in methanol (5 µg/ml) (Supplementary Table S1). Data acquisition and analysis were performed with MassHunter Workstation B 08.02 (Agilent Technologies, Santa Clara, USA).

The extracted DNA was enzymatically hydrolyzed and the aliquoted samples (10 μL typically containing 50 ng of digested DNA) were run in a reverse phase UPLC column (Eclipse C18 2.1 × 50 mm, 1.8 μm particle size, Agilent, Santa Clara, CA, USA) equilibrated and eluted (100 μL/min) with water/methanol/formic acid (95/5/0.1, all by volume). The effluent from the column was added to an electrospray ion source (Agilent Jet Stream) connected to a triple quadrupole mass spectrometer (Agilent 6460 QQQ, Santa Clara, CA, USA). The machine was operated in the positive ion multiple reaction monitoring mode using previously optimized conditions, and the intensity of specific MH+→fragment ion transitions were measured and recorded (5 mC m/z 242.1→126.1, 5hC 258.1→142.1 and dC m/z 228.1→112.1). The measured percentage of 5 mC in each experimental sample was calculated from the MRM peak area divided by the combined peak areas for 5 mC plus 5hmC plus C (total cytosine pool).

DCF was quantified on a 1200 infinity series liquid chromatography (Agilent, Waldbronn, Germany) coupled to triple quadrupole mass spectrometry (model 6460, Agilent) (LC-MSMS) with electron spray ionization (Jet Stream, Agilent) using a Kinetex™ C18 reverse phase column (2.1 × 100 mm, 1.7 U, 100 Å, Phenomenex, Aschaffenburg, Germany). The LC-MSMS settings and protocol were detailed by Esterhuizen-Londt et al. (2017 (link)) with a 0.5 pg on column (S/N > 5) limit of quantification. Prior to analysis, all samples were centrifuged at 10,000 × g at 10 °C for 30 min.

Data were acquired using an Agilent 6550 QToF instrument. Samples were ionized using an Agilent Jet Stream electrospray ionization source operated in negative mode. Gas temperature in the ion source was 150 °C with a flow rate of 14 L/min. Nebulizer pressure was 45 psig; sheath temperature was 325 °C with a gas flow rate of 12 L/min. Voltage for both capillary and nozzle was 2000 V. The funnel DC voltage was −30 V, funnel voltage drops were −100 and −50 V in the high- and low-pressure funnels, respectively. The RF voltages were 110 and 60 V in the high- and low-pressure funnels respectively. Mass spectra were acquired between 50 m/z and 1100 m/z with a rate of 2 spectra per second. Mass lock mixture was used as described in Wan et al.^{63 (link)}.

Compounds were separated on an Ascentis Express C18 HPLC column (100 × 3 mm, 2.7 µm) equipped with a C18 guard column (4 mm × 3 mm, 5 µm) Merck-Sigma group (Schnelldorf, Germany). The binary gradient elution mode was applied with solvent A containing 5 mM ammonium formate in water (pH 8.3) and solvent B containing methanol. The mobile phase gradient consisted of 5% B at 0 min; 5% B at 0.5 min; 40% B at 3.0 min; 100% B at 15 min; 100% B at 19 min; 5% B at 19.1 min; 5% B at 26.0 min; flow rate was set to 0.5 mL/min. The column thermostat and autosampler were maintained at 39 °C and at 18 °C, respectively. The injection volume was 2.0 µL. Compounds were detected using positive/negative ionization mode and dynamic multiple reaction monitoring (dMRM) scan mode. Ion transitions for 295 compounds are presented in Supplementary Table S2. The MRM time window was 60 s, and the cycle time was 1000 ms. The Agilent Jet Stream ion source parameters were as follows: drying gas temperature, 300 °C; sheath gas temperature, 350 °C; nebulizer, 35 psi; gas flow, 7 L/min; sheath gas flow, 11 L/min; capillary voltage, ± 3500 V; and nozzle voltage, +0, −1000 V. The HPLC effluent was directed into waste from 0 to 2.0 min.