To date, there are 56 tRNA sequences derived from S. cerevisiae in the T-psi-C database, a collection of tRNA sequences and their modifications obtained from high throughput tRNA sequencing (see a histogram summarizing the mass distribution of the isoforms in Figure ). This set of sequences is used for generation of theoretical precursor and fragment ion isotopic distributions. The heterogeneity of the desalted tRNA extract is significant, as illustrated in the MS1 spectrum in Figure . There are multiple overlapping tRNA isoforms throughout the MS1 spectrum, as shown in Figure and with expanded detail in Figure for the m/z range spanning 849 to 887, making it challenging to assign compositions of precursors with confidence. A set of isolation windows, each 2 m/z wide, is overlaid on the MS1 spectrum in Figure , indicating the windows that are selected for isolation and subsequent UV photoactivation to promote charge reduction of the most abundant precursor ions between m/z 849-887. In this manner, precursor ion isotopic distributions corresponding to each of the 56 yeast tRNA sequences recorded in the T-Psi-C database were identified with or without possible 3' terminal adenosine loss, totaling 112 sequences. In total, 122 sets of theoretical fragment ions based on identified tRNA precursor ions and their corresponding charge states, some of which were identified in more than one charge state, were generated from this set of 112 sequences. Here, CAD (30 NCE) was applied to the precursor ions isolated between m/z 883.5-885.5, corresponding to the second isolation window from the right shown in Figure . This window contains the Asp GUC species identified in the UVPD spectrum of the same isolation window (Figure ). Fragment ions generated from the database sequence (without a 3' terminal adenosine) were identified directly in the m/z domain, resulting in the sequence coverage map shown in Figure . A sequence coverage of 65.8% was achieved for this tRNA, resulting in a significant P-score of 1.30e-122, indicative of high confidence in identification of the tRNA. Moreover, fragment ions -mainly c and y fragments -bracket many residues near the 5' and 3' termini, respectively, localizing several covalent modifications present in the predicted sequence. These modifications include number of co-isolated species in same charge state (26-) with substantial overlap in the m/z domain. Theoretical isotope distributions of potential precursor identifications for six tRNAs are overlaid on the array of charge-reduced precursors in Figure , resulting in six potential tRNA identities (accession tdbR00000362, 370, 443, 444, 454, and 555). Although not all match the observed isotopic abundances, all predicted isotopes match within a 10 ppm m/z tolerance of the centroided m/z values. Additional precursors at m/z values (e.g., m/z 906-907) in Figure were unmatched, which may be indicative of additional tRNA isoforms that are yet uncharacterized.