As part of RNA characterization, the identification of post-transcriptional modifications can be performed using hyphenation of separation methods with mass spectrometry. To identify RNA modifications with those methods, a first total digestion followed by a dephosphorylation step are usually required to reduce RNA to nucleosides. Even though effective digestion and dephosphorylation are essential to avoid further complications in analysis and data interpretation, to our knowledge, no standard protocol is yet referenced in the literature. Therefore, the aim of this work is to optimize the dephosphorylation step using a total extract of transfer RNA (tRNA)¹ from B. taurus as a model and to determine and fix two protocols, leading to complete dephosphorylation, based on time and bacterial alkaline phosphatase (BAP)² consumptions. Capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) was used to estimate the dephosphorylation efficiency of both protocols on many canonical and modified nucleotides. For a timesaving protocol, we established that full dephosphorylation was obtained after a 4-hour incubation at 37 °C with 7.5 U of BAP for 1 µg of tRNA. And for a BAP-saving protocol, we established that full dephosphorylation was obtained 3.0 U of BAP after an overnight incubation at 37 °C. Both protocols are suitable for quantitative analyses as no loss of analytes is expected. Moreover, they can be widely used for all other RNA classes, including messenger RNA or ribosomal RNA.

作为 RNA 表征的一部分，转录后修饰的鉴定可以使用质谱分离方法的连字符进行。为了用这些方法鉴定 RNA 修饰，通常需要先进行全消化，然后进行去磷酸化步骤，以将 RNA 还原为核苷。尽管有效的消化和去磷酸化对于避免分析和数据解释的进一步复杂化是必不可少的，但据我们所知，文献中尚未引用标准协议。因此，这项工作的目的是使用来自B. taurus的转移 RNA (tRNA) ^{1的总提取物优化去磷酸化步骤}作为模型，并根据时间和细菌碱性磷酸酶 (BAP) ^{2确定和修复两个方案，从而导致完全去磷酸化}消费。毛细管电泳串联质谱 (CE-MS/MS) 用于估计两种协议对许多规范和修饰核苷酸的去磷酸化效率。对于节省时间的方案，我们确定在 37 °C 下用 7.5 U BAP 孵育 4 小时后，1 µg tRNA 可完全去磷酸化。对于节省 BAP 的方案，我们确定在 37°C 下过夜孵育后，3.0 U 的 BAP 获得了完全去磷酸化。两种协议都适用于定量分析，因为预计不会损失分析物。此外，它们可广泛用于所有其他 RNA 类别，包括信使 RNA 或核糖体 RNA。