Figure 3

Direct detection of 5-methylcytidine and 5-hydroxymethylcytidine in Oxytricha trifallax DNA using high-resolution nano-flow UPLC-mass spectrometry. Nucleosides generated from purified O. trifallax DNA isolated 0 h, 36 h, 40 h and 64 h post-conjugation, or from standards (synthetic PCR products containing either unmodified nucleosides, 5-methylcytidine or 5-hydroxymethylcytidine), were subjected to LC-MS on a high-resolution nano-flow UPLC - Orbitrap mass spectrometry platform. Extracted chromatograms of the (a) cytidine, (b) 5-methylcytidine and (c) 5-hydroxymethylcytidine [M+H]⁺ ions are shown, displaying a single prominent peak for each molecular species across the chromatographic timescale. Inset into the chromatograms are mass spectra of the detected [M+H]⁺ ion for each molecular species; empirical mass measurements for these ions were each within ±0.0005 Da of theoretical values for cytidine, 5-methylcytidine and 5-hydroxymethylcytidine atomic compositions.