DNA methylation is a critical epigenetic mark in mammalian cells. Many aspects of DNA methylation maintenance have been characterized; however, the exact kinetics of post-replicative methylation maintenance remain a subject of debate. Here we develop isolation of DNA by 5-ethynyl-deoxyuridine labelling for mass spectrometry (iDEMS), a highly sensitive, quantitative mass spectrometry-based method for measuring DNA modifications on metabolically labelled DNA. iDEMS reveals an unexpectedly hemi-methylated landscape on nascent DNA. Combining iDEMS with metabolic labelling reveals that methylation maintenance is outpaced by cell division in mouse embryonic stem cells. Our approach shows that hydroxymethylation is perpetually asymmetric between sister strands in favour of the parental, template strand. iDEMS can be coupled with immunoprecipitation of chromatin proteins, revealing features of DNA methylation–histone modification crosstalk and suggesting a model for interplay between methylation and nucleosome assembly. iDEMS therefore elucidates long-standing questions about DNA modification propagation and provides an important orthogonal technology to understanding this process in dynamic cellular contexts.

DNA 甲基化是哺乳动物细胞中一个重要的表观遗传标记。DNA 甲基化维持的许多方面已被表征；然而，复制后甲基化维持的确切动力学仍然存在争议。在这里，我们通过用于质谱法 (iDEMS) 的 5-乙炔基-脱氧尿苷标记分离 DNA，这是一种高度灵敏、基于定量质谱法的方法，用于测量代谢标记 DNA 上的 DNA 修饰。iDEMS 揭示了新生 DNA 上出乎意料的半甲基化景观。将 iDEMS 与代谢标记相结合表明，小鼠胚胎干细胞中的细胞分裂超过了甲基化维持。我们的方法表明，羟甲基化在姐妹链之间永远是不对称的，有利于亲本模板链。iDEMS 可以与染色质蛋白的免疫沉淀相结合，揭示 DNA 甲基化-组蛋白修饰串扰的特征，并提出甲基化和核小体组装之间相互作用的模型。因此，iDEMS 阐明了有关 DNA 修饰传播的长期问题，并提供了一种重要的正交技术来理解动态细胞环境中的这一过程。