SpectrSeqTools: LC-MS/MS-Based De Novo Sequencing of Small Ribonucleic Acids with Post-Transcriptional Modifications

Abstract Post-transcriptional RNA modifications are key regulators of fundamental cellular processes. Yet, their precise biological functions remain largely unresolved. A major challenge is their reliable detection. While sequencing-based approaches are limited to a small subset of RNA modifications, mass spectrometry has the potential to directly identify various modified nucleotides within their sequence context. However, existing tools for nucleic acid analysis offer severely constrained functionality. Here, we introduce a set of algorithmic innovations implemented in SpectrSeqTools, an open-source LC-MS/MS-based software platform that enables fully automated de novo RNA sequencing together with comprehensive modification profiling of short RNA fragments directly from raw data. We further validated prediction accuracy and robustness using experimental and simulated data. As such, SpectrSeqTools effectively broadens the scope of nucleic acid mass spectrometry, enabling the read-out of up to 144 RNA modifications within their sequence context.