Sauvage Center for Molecular Sciences, Department of Chemistry, Wuhan University, Wuhan, China.
Methods Mol Biol. 2021;2298:247-259. doi: 10.1007/978-1-0716-1374-0_16.
The recent discovery of reversible chemical modifications on mRNA has opened a new era of post-transcriptional gene regulation in eukaryotes. Among these modifications identified in eukaryotic mRNA, N7-methylguanosine (mG) is unique owing to its presence in the 5' cap structure. Recently, it has been reported that mG also exists internally in mRNA. Here, we describe a protocol of combining differential enzymatic digestion with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis to detect internal mG modification in mRNA. This protocol can also be used to quantify the level of mG at both the 5' cap and internal positions of mRNA.
最近在 mRNA 上发现的可逆化学修饰为真核生物中的转录后基因调控开辟了一个新时代。在真核 mRNA 中鉴定出的这些修饰中，N7-甲基鸟苷（mG）是独特的，因为它存在于 5'帽结构中。最近，据报道 mG 也存在于 mRNA 内部。在这里，我们描述了一种结合差示酶消化与液相色谱-电喷雾电离-串联质谱（LC-ESI-MS/MS）分析来检测 mRNA 内部 mG 修饰的方案。该方案还可用于定量测定 mRNA 5'帽和内部位置 mG 的水平。
