Transfer RNAs (tRNAs) are the most heavily modified RNA species. Liquid chromatography coupled with mass spectrometry (LC–MS/MS) is a powerful tool for characterizing tRNA modifications, which involves pretreating tRNAs with base-specific ribonucleases to produce smaller oligonucleotides amenable to MS. However, the quality and quantity of products from base-specific digestions are severely impacted by the base composition of tRNAs. This often leads to a loss of sequence information. Here, we report a method for the full-range profiling of tRNA modifications at single-base resolution by combining site-specific RNase H digestion with the LC–MS/MS and RNA-seq techniques. The key steps were designed to generate high-quality products of optimal lengths and ionization properties. A linear correlation between collision energies and the m/z of oligonucleotides significantly improved the information content of collision-induced dissociation (CID) spectra. False positives were eliminated by up to 95% using novel inclusion criteria for collecting a census of modifications. This method is illustrated by the mapping of mouse mitochondrial tRNAHis(GUG) and tRNAVal(UAC), which were hitherto not investigated. The identities and locations of the five species of modifications on these tRNAs were fully characterized. This approach is universally applicable to any tRNA species and provides an experimentally realizable pathway to the de novo sequencing of post-transcriptionally modified tRNAs with high sequence coverage.

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