High-throughput profiling of RNA modifications by ultra-performance liquid chromatography coupled to complementary mass spectrometry: Methods, quality control, and applications

Although next-generation sequencing technology has been used to delineate RNA modifications in recent years, the paucity of appropriate converting reactions or specific antibodies impedes the accurate characterization and quantification of numerous RNA modifications, especially when these modifications demonstrate wide variations across developmental stages and cell types. In this study, we developed a high-throughput analytical platform coupling ultra-performance liquid chromatograph (UPLC) with complementary mass spectrometry (MS) to identify and quantify RNA modifications in both synthetic and biological samples. Sixty-four types of RNA modifications, including positional isomers and hypermodified ribonucleosides, were successfully monitored within a 16-min single run of UPLC–MS. Two independent methods to cross-validate the purity of RNA extracted from Caenorhabditis elegans (C. elegans) were developed using the coexisting C. elegans and Escherichia coli (E. coli) as a surveillance system. To test the validity of the method, we investigated the RNA modification landscape of three model organisms, C. elegans, E. coli, and Arabidopsis thaliana (A. thaliana). Both the identity and molarity of modified ribonucleosides markedly varied among the species. Moreover, our platform is not only useful for exploring the dynamics of RNA modifications in response to environmental cues (e.g., cold shock) but can also help with the identification of RNA-modifying enzymes in genetic studies. Cumulatively, our method presents a novel platform for the comprehensive analysis of RNA modifications, which will be of benefit to both analytical chemists involved in biomarker discovery and biologists conducting functional studies of RNA modifications.