Likewise, specific fragment ions like the identified z66 5-and y63 5-ions display nearly identical isotopic distributions; however, the observed ion of m/z 762 is likely attributable to the Val IAC tRNA (tdbR00000464) rather than Tyr GPA tRNA (tdbR00000555) based on the more confident precursor identification in 4A, additional confident fragment assignments in 4B, and higher overall abundances of identified fragment ions in 4B compared to the alternative Tyr GPA tRNA (tdbR00000555). Figure displays a summary of fragment identification results from fragment ion searches undertaken for the six precursors. Sequence coverage maps and the corresponding P-scores for the identified fragments of each tRNA are displayed in Figures . A summary of sequence coverage versus calculated P-score is displayed in Figure for all 376 searches. Sequence coverages and P-scores for these 376 searches can be found in the Supporting Information. To assess the quality of top-down annotation for this highly heterogenous tRNA sample, decoy searches were carried out by performing a fragment ion search of each CAD spectrum for a tRNA that was not identified as a charge-reduced precursor in the corresponding UVPD spectrum of that same isolation window. The outcomes of these searches, displayed in red in Figure , allowed generation of a cutoff P-score, where only tRNAs exhibiting higher P-scores were considered accurate identifications based on their fragment ions. This strategy was more stringent than a traditional scrambled sequence search, owing to the highly heterogeneous MS/MS spectra, the high sequence homology across tRNAs -particularly at their 3' termini -and the potential for differential modification of tRNAs to influence the species present in a given spectrum, which may summarizing characterization of tRNAs across the 376 fragment ion searches undertaken. Decoy searches (red) were carried out on each CAD spectrum (50 total), and P-scores of experimental tRNA fragment identifications (blue) were compared to the P-score of the decoy (red). Validated spectra with more confident P-scores compared to the decoys are shown in bold blue; those with P-scores less confident than the decoys are gray. Of the 376 fragment ion searches, 174 exhibited higher Pscores than the corresponding decoy search of the same CAD spectrum, and the average sequence coverage among these is 46.1%.