Messenger RNA (mRNA) can be sequenced via indirect approaches such as Sanger sequencing and next generation sequencing (NGS), or direct approaches like bottom-up mass spectrometry (MS). Direct sequencing allows the confirmation of RNA modifications. However, the conventional bottom-up MS approach involves time-consuming in-solution digestions that require a large amount of sample, and can lead to the RNase contamination of the LC-MS system and column. Here, we describe a platform that enables online nucleotide mapping of mRNAs via the use of immobilized RNase cartridges and 2D-LC-MS instrumentation. The online approach was compared to conventional offline digestion protocols adapted from two published studies. For this purpose, five model mRNAs of varying lengths (996–4521 nucleotides) and chemistries (unmodified uridine vs 5-methoxyuridine (5moU)) were analyzed. The profiles and sequence coverages obtained after RNase T1 digestion were discussed. The online nucleotide mapping achieved comparable or slightly greater sequence coverage for the 5 mRNAs (5.8–51.5%) in comparison to offline approaches (3.7–50.4%). The sequence coverage was increased to 65.6–85.6 and 69.7–85.0% when accounting for the presence of nonunique digestion products generated by the RNase T1 and A, respectively. The online nucleotide mapping significantly reduced the digestion time (from 15 to <5 min), increased the signal intensity by more than 10-fold in comparison to offline approaches.

中文翻译：

---

mRNA 在线核苷酸图谱


信使 RNA (mRNA) 可以通过桑格测序和下一代测序 (NGS) 等间接方法或自下而上质谱 (MS) 等直接方法进行测序。直接测序可以确认 RNA 修饰。然而，传统的自下而上 MS 方法涉及耗时的溶液内消化，需要大量样品，并且可能导致 LC-MS 系统和色谱柱的 RNase 污染。在这里，我们描述了一个平台，该平台可以通过使用固定化 RNase 盒和 2D-​​LC-MS 仪器来实现 mRNA 的在线核苷酸图谱。将在线方法与改编自两项已发表研究的传统离线消化方案进行了比较。为此，我们分析了 5 个不同长度（996-4521 个核苷酸）和化学成分（未修饰尿苷与 5-甲氧基尿苷 (5moU)）的 mRNA 模型。讨论了 RNase T1 消化后获得的谱图和序列覆盖率。与离线方法 (3.7–50.4%) 相比，在线核苷酸作图对 5 种 mRNA (5.8–51.5%) 实现了相当或稍高的序列覆盖率。当考虑到 RNase T1 和 A 产生的非独特消化产物时，序列覆盖率分别增加到 65.6-85.6 和 69.7-85.0%。与离线方法相比，在线核苷酸图谱显着缩短了消化时间（从 15 分钟缩短至 <5 分钟），信号强度增加了 10 倍以上。