LC-MS quantification of 5mC and 5hmC nucleosides

Purified genomic DNA was hydrolyzed enzymatically as described previously⁷⁵. In brief, DNA was digested by nuclease P1 (Sigma, Cat#N8630) in the presence of 0.2 mM ZnSO₄ and 20 mM NaAc (pH 5.3) at 55 °C for at least 1 h and then was dephosphorylated with calf intestinal alkaline phosphatase (CIAP, Takara, Cat#2250A) at 37 °C for an additional 3 h. The samples were cleaned up by spin dialysis (Nanosep with 10K omega, PALL). The nucleosides were resolved by HPLC (Agilent 1260) as previously described⁷⁶. In brief, a Hypersil Gold aQ column (Thermo Fisher Scientific, 100 mm; particle size, 1.9 μm) was used with a gradient elution starting with 0.1% (v/v) formic acid in water (A) and adding acetonitrile (LC-MS grade) with 0.1% (v/v) formic acid (B) at 25 °C. 0-5 min: 100% A, 5–12.5 min: 100% A to 88% A; 12.5–14 min: to 0 % A; 14–16 min: 100% A, 16–19 min: 100% A. The HPLC eluent was analyzed on an Agilent 6495 triple quadrupole mass spectrometer using multiple-reaction monitoring in positive ion mode. Transitions for each nucleoside: deoxycytidine (dC): 228.1→112.1 m/z, dG (Sigma, Cat#D7145), 5mdC: 242.1→126.1 m/z, 5hmdC: 258.1→142.1. A detailed procedure and instrumentation for the LC-MS/MS analyses are described previously⁷⁶. For quantifications, a series of concentrations of nucleoside standards, were run for every batch of experiments to obtain their corresponding standard curves. dG (Sigma, Cat#D7145), dC (Sigma, Cat#D3897), 5mdC (Cayman, Cat#16166), 5hmdC (Berry & Associates, Cat#PY-7588). The ratios of 5mdC/dG and 5hmdC/C, were subsequently calculated. Data were analyzed using Microsoft Excel and GraphPad Prism 7 software.