Cyclic dinucleotides (CDNs) are key secondary messenger molecules produced by cyclic dinucleotide synthases that trigger various cellular signaling cascades from bacteria to vertebrates. In mammals, cyclic GMP-AMP synthase (cGAS) has been shown to bind to intracellular DNA and catalyze the production of the dinucleotide 2′3′ cGAMP, which signals downstream effectors to regulate immune function, interferon signaling, and the antiviral response. Despite the importance of CDNs, sensitive and accurate methods to measure their levels in vivo are lacking. Here, we report a novel LC-MS/MS method to quantify CDNs in vivo. We characterized the mass spectrometric behavior of four different biologically relevant CDNs (c-di-AMP, c-di-GMP, 3′3′ cGAMP, 2′3′ cGAMP) and provided a means of visually representing fragmentation resulting from collision-induced dissociation at different energies using collision energy breakdown graphs. We then validated the method and quantified CDNs in two in vivo systems, the bacteria Escherichia coli OP50 and the killifish Nothobranchius furzeri. We found that optimization of LC-MS/MS parameters is crucial to sensitivity and accuracy. These technical advances should help illuminate physiological and pathological roles of these CDNs in in vivo settings.

环状二核苷酸 (CDN) 是由环状二核苷酸合酶产生的关键二级信使分子，可触发从细菌到脊椎动物的各种细胞信号级联反应。在哺乳动物中，已显示环状 GMP-AMP 合酶 (cGAS) 与细胞内 DNA 结合并催化二核苷酸 2'3' cGAMP 的产生，该二核苷酸向下游效应器发出信号以调节免疫功能、干扰素信号传导和抗病毒反应。尽管 CDN 很重要，但缺乏灵敏和准确的方法来测量其体内水平。在这里，我们报告了一种新的 LC-MS/MS 方法来量化体内 CDN。我们表征了四种不同的生物学相关 CDN（c-di-AMP、c-di-GMP、3'3' cGAMP、2'3' cGAMP）并提供了一种使用碰撞能量分解图直观地表示由不同能量下的碰撞诱导解离产生的碎片的方法。然后，我们在两个体内系统（细菌）中验证了该方法并量化了 CDN大肠杆菌OP50 和鳉鱼 Nothobranchius furzeri。我们发现优化 LC-MS/MS 参数对灵敏度和准确性至关重要。这些技术进步应该有助于阐明这些 CDN 在体内环境中的生理和病理作用。