Standard-free Quantitative Mass Spectrometry of RNA Modifications

The coupling of liquid chromatography (LC) to mass spectrometry (MS) is an accurate and proven technique for identifying different types of RNA modifications. However, due to the lack of standards and analytical biases, epitranscriptome-wide quantitative MS analysis of RNA modifications is challenging. Here, we report the development of a standard-free quantitative mass spectrometry (SqMS) method for measuring multiple RNA modifications. Starting from the extraction of total RNA to the LC-MS analysis of digested RNA, the commonly used approach is used in SqMS. To eliminate the biases that resulted from MS ionization efficiency, the SqMS method adjusts the MS signal of each ribonucleoside with a corresponding factor. Each adjustment factor is derived from measuring a serially diluted control sample, which contains a comparable set of RNA modifications as in our samples of interest. By using a post-column UV detector and the information on molar absorptivity, the ribonucleoside concentration in each diluted control sample is determined and plotted against the corresponding MS signal. The resulting dilution curve of each detectable ribonucleoside is equivalent to the standard curve, thus no ribonucleoside standard is required. After normalizing the adjusted MS signals, the accuracy of SqMS in the quantitation of multiple RNA modifications within the same sample is as good as performing the absolute quantitation. By using SqMS, the epitranscriptomic variations in glioblastoma cells were accurately differentiated.