Mass-Spectrometry-Based Assay at Single-Base Resolution for Simultaneously Detecting m⁶A and m⁶Am in RNA

The methylation modifications of adenosine, especially N⁶-methyladenosine (m⁶A) and N⁶, 2'-odimethyladenosine (m⁶Am), play vital roles in various biological, physiological, and pathological processes. However, current methods for detecting these modifications at single-base resolution have limitations. Mass spectrometry (MS), a highly accurate and sensitive technique, can be utilized to differentiate between m⁶A and m⁶Am by analyzing the molecular weight differences in their fragments during tandem MS analysis. In this study, we present an MS-based method that allows for the simultaneous determination of m⁶A and m⁶Am sites in targeted RNA fragments at single-nucleotide resolution. The approach involves the utilization of tandem MS in conjunction with targeted RNA enrichment and enzymatic digestion, eliminating the need for PCR amplification. By employing this strategy, we can accurately identify m⁶A and m⁶Am sites in targeted RNA fragments with high confidence. To evaluate the effectiveness of our method, we applied it to detect m⁶A and m⁶Am sites in cell and tissue samples. Furthermore, we verified the accuracy of our approach by performing CRISPR/Cas9-mediated knockout of the corresponding methyltransferases. Overall, our MS-based method offers a reliable and precise means for the simultaneous detection of m⁶A and m⁶Am modifications in targeted RNA fragments, providing valuable insights into the functional characterization of these modifications in various biological contexts.