Mass Spectrometry Analysis of 5hmC Levels

SKM-1 cells were cultured with 300 µM ascorbate or phosphoascorbate in 12-well plates at 0.4 x 10⁶ cells/ml in 2 ml per well for periods up to 4 days. The medium was refreshed by diluting 1:5 in fresh media with the addition of 300 µM ascorbate or phosphoascorbate after 2 days. At the end of the incubation period, the cells were harvested and the DNA was extracted using a DNA extraction kit (DNeasy Blood and Tissue Kit Cat No. 69504, Qiagen, Hilden, Germany).

A stable isotope dilution LC-MS/MS method was used for the detection and quantification of 2’-deoxycytidine, 5-methyl-2’-deoxycytidine and 5-hydroxymethyl-2’-deoxycytidine. Isotopically labeled standards [2’-deoxycytidine (¹³C, ¹⁵N₂), 5-methyl-2’-deoxycytidine (¹³C, ¹⁵N₂) and 5-hydroxy-methyl-2’-deoxycytidine (d₃)] were used to control for experimental variations such as recovery, matrix effect, and ionization. Standard calibration curves using the ratio of light to heavy isotopes were used for quantification. One µg of SKM-1 DNA was hydrolyzed using a nucleoside digestion kit M0649S (New England Biolabs, Ipswich, MA, USA) in the presence of internal standards [130 fmoles 2’-deoxycytidine (¹³C, ¹⁵N₂), 5 fmoles 5-methyl-2’-deoxycytidine (¹³C, ¹⁵N₂) and 0.013 fmoles 5-hydroxy-methyl-2’-deoxycytidine (d₃)].

Standards and digested SKM-1 DNA samples were analyzed using a 6500 QTrap mass spectrometer (Sciex, Framingham, MA, USA) coupled to an Infinity 1290 LC system (Agilent, Santa Clara, CA, USA). Standards and samples were stored on the autosampler tray at 5°C. An Acclaim RSLC Polar Advantage II 120Å column (150 x 2.1 mm, Thermo Fisher Scientific Inc., Waltham, MA, USA) was used for chromatographic separation using 100% water (0.1% formic acid) as Solvent A and 100% acetonitrile (0.1% formic acid) as Solvent B. A flow rate of 0.2 mL/minute was used. The column temperature was set to 40°C. The analytes were eluted during the initial isocratic phase with 100% Solvent A over 3.5 minutes. The column was then flushed with 5% Solvent A and 95% Solvent B for 2.5 minutes, and then re-equilibrated at initial conditions for 5 minutes. Data were analyzed using Analyst 1.7.1 (Sciex, Framingham, MA, USA). All species were quantified by fragmenting the singly-charged parent ion [M+H]⁺, monitoring the fragment ion resulting from the loss of the deoxyribose sugar in positive-ion mode (Table 1), and measuring the area under the curve of the resulting peak (Fit: Linear, Weighting: None, Regression Parameter: Area). The concentration of deoxycytidine, 5mC and 5hmC in each sample was calculated by relating the peak area ratio of the light to the heavy isotope to standard calibration curves, and then converted to a percentage of the total cytidine species. The assay was validated by measuring the relative composition of cytidine species in frontal cortex and liver tissues (Dunkin Hartley guinea pigs) for comparison. For guinea pig tissue samples, one µg of DNA was hydrolyzed in the presence of 340 fmoles 2’-deoxycytidine (¹³C, ¹⁵N₂), 10 fmoles 5-methyl-2’-deoxycytidine (¹³C, ¹⁵N₂) and 1.3 fmoles 5-hydroxy-methyl-2’-deoxycytidine (d₃)).

The m/z values for the singly-charged parent and fragment ions and the optimised parameters that were used to quantify each analyte in LC-MS/MS experiments.

DP, declustering potential; EP, entrance potential; CE, collision energy; CXP, cell exit potential.