Global Analysis of DNA Methylation by LC-MS

DNA samples (1 or 2 μg) were digested with Degradase Plus (Zymo Research) at a ratio of 5 U/μg in a final volume of 25 μl overnight at 37°C, then diluted to 100 μl by adding 75 μl 0.1% (v/v) formic acid (ROTIPURAN^®, Carl Roth) in ultrapure water (Milli-Q^® Advantage A10 water purification system, Merck Millipore) (Capuano et al., 2014). Calibration curves were prepared by dissolving 2′-deoxycytidine (dC, Sigma-Aldrich) and 5-methyl-2′-deoxycytidine (5mdC, Cayman Chemical) in nuclease-free water on ice, each to a final concentration of 1 mg/ml. The nucleoside stock solutions were diluted with 0.5% (v/v) formic acid in ultrapure water to yield 1, 2.5, 5, 10, 100, 250, 500, 1000, and 2000 pg/μl dC/^5mdC-standard solutions. The analysis of genomic DNA was carried out by injecting 5-μl digested DNA samples and standard solutions into an UltiMate 3000 HPLC system (Dionex) followed by quantification in an amazon EDT ion trap mass spectrometer (Bruker Daltonics). Components were separated on a reversed-phase column (Kinetex C18, 2.6 μm, 50 × 2.1 mm, 100 Å, Phenomenex) under isocratic conditions [0.1% (v/v) formic acid (ROTIPURAN) and 5% (v/v) acetonitrile (ROTISOLV, Carl Roth) in ultrapure water] at a flow rate of 150 μl/min and 30°C. Cytidine residues were quantified by multiple reaction monitoring (MRM) after positive electrospray ionization using the following ion source parameters: 1.0 bar nebulizer pressure, 8 l/min drying gas, 200°C drying temperature, 4500 V capillary power and 500 V end-plate offset. Ionization and MRM conditions were optimized for fragmentation reactions for mass/charge ratios 228.1→112.0 (for dC) and 242.1→126.1 (for 5mdC). The data were analyzed using Compass Data Analysis v4.2 (Bruker Daltonics). Fold changes in relative global DNA methylation levels were calculated for treatment groups exposed to bacteria against the corresponding control groups.