(T0930-03-18) Accurate Quantification of DNA Methylation and Hydroxymethylation in Biosamples Using Liquid Chromatography–Tandem Mass Spectrometry

Purpose: DNA methylation and hydroxymethylation are crucial epigenetic modifications involved in gene expression regulation and genome stability, and they are closely linked to cancer development and progression. Conventional methylation assays, such as bisulfite conversion or antibody-based ELISA, are limited by low specificity, labor intensity, and an inability to distinguish between methylation and hydroxymethylation. To address these limitations, we developed a sensitive, reproducible, and specific LC-MS/MS assay for simultaneous quantification of 2′-deoxycytidine (2-dC), 5-methyl-2′-deoxycytidine (5-mdC), and 5-hydroxymethyl-2′-deoxycytidine (5-hmdC). The method employs stable isotope-labeled internal standards and was applied to analyze DNA extracted from mouse liver tissue.

Methods: Stock solutions of the three analytes were prepared at 1 mg/mL, and working standards were prepared in water at ranges of 50–50,000 ng/mL (2-dC), 5–5,000 ng/mL (5-mdC), and 0.5–100 ng/mL (5-hmdC). Isotope-labeled standards included 2′-deoxycytidine-¹³C,¹⁵N, 5-methyl-2′-deoxycytidine-d₃, and 5-hydroxymethyl-2′-deoxycytidine-d₃. Samples were filtered through Amicon Ultra filters (10K NMWL) at 4°C, spiked with internal standards, and subjected to LC-MS/MS using a 6500+ Triple Quad system and a reverse-phase Atlantis T3 Waters C18 column (2.1 × 100 mm, 3 µm). Chromatographic separation employed a binary gradient system using 2 mM ammonium acetate with 0.04% acetic acid in water (A) and methanol (B), at 0.4 mL/min. The total run time was 9 minutes. Calibration curves were constructed using spiked negative control samples

Results: The method showed MRM transitions of m/z 228.154 → 112.1 for 2-dC, 242.143 → 126.2 for 5-mdC, and 258.135 → 142.1 for 5-hmdC. Quantification in liver DNA samples from eight female mice revealed concentrations ranging from 711–1010 ng/mL (2-dC), 22.7–40.1 ng/mL (5-mdC), and 0.688–2.15 ng/mL (5-hmdC). No analytes were detected in the negative control, indicating high specificity. Calibration curves were linear (R² > 0.999), and the intra-/inter-day precision and accuracy were within 10%. The assay demonstrated excellent compound stability and is currently being applied to various biological matrices including cell culture media.

Conclusion: This LC-MS/MS assay enables accurate, specific, and reproducible quantification of 2-dC, 5-mdC, and 5-hmdC in biosamples. Its application in mouse liver tissue highlights its potential utility for broader biomarker studies in clinical and research settings.

References: Xiong, J., Jiang, H.-P., Peng, C.-Y., Deng, Q.-Y., Lan, M.-D., Zeng, H., Zheng, F., Feng, Y.-Q., & Yuan, B.-F. (2015). DNA hydroxymethylation age of human blood determined by capillary hydrophilic-interaction liquid chromatography/mass spectrometry. Clinical Epigenetics, 7(72).
Németh, K., Mészáros, K., Szabó, B., Butz, H., Arányi, T., & Szabó, P. T. (2021). A relative quantitation method for measuring DNA methylation and hydroxymethylation using guanine as an internal standard. Analytical Methods, 13(36), 4614–4622.
Marchante‐Gayón, J. M., Carcelén, J. N., Rodríguez, H. P., Pineda‐Cevallos, D., Sánchez, L. R., González‐Gago, A., Rodríguez‐González, P., & García Alonso, J. I. (2024). Quantification of modified nucleotides and nucleosides by isotope dilution mass spectrometry. Mass Spectrometry Reviews, 43(5), 998–1018.

Acknowledgements: This study was supported by the Cancer Prevention & Research Institute of Texas (CPRIT) Core Facilities Support Awards (RP240430), NIH/NIMHD grant U54MD007605, CPRIT Individual Investigator Research Award (RP200081), and CPRIT Individual Investigator Research Awards for Prevention and Early Detection (RP230063).