More than 170 distinct chemical modifications have been identified in cellular RNAs. Nucleotide modifications influence RNA stability, structure, localisation, translation, and interactions with RNA-binding proteins. The functional versatility of modified RNAs plays critical roles in processes such as embryonic development, stress response, and innate immunity. Accordingly, aberrant modification patterns have been linked to cancer, neurological disorders, and viral infections. Despite the growing interest in the epitranscriptome, the quantification of RNA modifications remained challenging, especially beyond the few modifications that are extensively studied. There is a pressing need for sensitive and high-throughput analytical methods to quantify nucleoside modifications.
To address these challenges, we adapted workflows for the targeted mass spectrometry identification of modified nucleosides from bulk RNA, using Liquid Chromatography coupled with a Triple Quadrupole Mass Spectrometer (LC-TQMS). We demonstrate the quantification of RNA modifications that are otherwise challenging for detection by conventional approaches. These include the quantification of 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U) in bulk RNA from S. cerevisiae, validated by the knockout of the thiolation enzyme Urm1 that is required for 2-thiouridine (s²U) modification. Using the same mass spectrometry workflow, we quantified other RNA and DNA modifications, such as N6-methyladenosine (m⁶A), pseudouridine (Ψ), and 5-methylcytidine (m⁵C), among others. Through conjugation with RNA isolation protocols, we demonstrate the detection and quantification of bulk RNA modification profiles across distinct RNA classes in yeast and human cells, while comparing different cell states, such as cell differentiation. The results highlight the versatility of the method in terms of both the sample variability and the detected modifications, providing a unified workflow for discovery and mechanistic studies of RNA processing and modifications.