通过同位素稀释LC-MS/MS同时量化cytidine,methylcytidine和hydroxymethylcytidine,并应用于小鼠肝脏样本
Pooja Mishra1, Jing Ma1, Huan Xie1
1Department of Pharmaceutical Sciences, Texas Southern University, 3100 Cleburne Street, Houston, TX 77004, USA.
Journal of chromatography. A
|January 25, 2026
概括
这项研究引入了一种新的LC-MS/MS方法,用于精确测量DNA甲基化和基甲基化. 该试验准确量化了这些表观遗传标记,揭示了老化随着老鼠肝脏的氧甲基化增加.
科学领域:
- 表观遗传学 在表观遗传学中,表观遗传学是指表观遗传学.
- 分子生物学分子生物学
- 分析化学 分析化学
背景情况:
- 基因甲基化和基甲基化是影响基因表达,基因组稳定性,癌症和衰老的关键表观遗传修饰.
- 现有的方法,如二硫酸盐转化或ELISA缺乏特异性,无法区分甲基化和基甲基化.
- 这些局限性要求开发精确的分析技术,以准确量化.
研究的目的:
- 开发和验证一种敏感,可复制和特定的液体染色学-并联质谱法 (LC-MS/MS) 方法.
- 为了同时量化5-甲基-2'-脱氧基丁 (5-mdC) 和5-基甲基-2'-脱氧基丁 (5-hmdC) 以及2'-脱氧基丁 (2-dC).
- 评估该方法在分析生物样本中的表观遗传变化的适用性,例如老化相关的小鼠肝脏修饰.
主要方法:
- 开发一种新的LC-MS/MS测定方法,使用稳定同位素标记的5-mdC,5-hmdC和2-dC的内部标准.
- 从小鼠肝脏样本中净化和酶化消化DNA,然后进行过和添加内部标准.
- 使用SCIEX 6500+ Triple Quad LC-MS/MS系统与Atlantis T3 C18柱和优化的二进制梯度化进行分析.
主要成果:
- LC-MS/MS方法证明了对目标分析物的出色染色学分离和特异性.
- 在2-dC,5-mdC和5-hmdC的广泛校准范围内实现了高线性 (R2 > 0.999).
- 在雌性小鼠肝脏中观察到5-hmdC水平的显著年龄相关增加 (0.0958%至0.1984%),而5-mdC水平保持稳定.
结论:
- 开发的LC-MS/MS测定方法提供了组织样本中DNA甲基化和基甲基化的准确和可重复量化.
- 该方法的特异性和灵敏性克服了传统分析的局限性.
- 这种测试在细胞培养研究中具有广泛的潜在应用,并在临床研究中作为衰老和癌症的生物标志物.
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