In the representative CID spectrum shown in Figure for the tRNA-Phe isoform, there was an unusually high abundance w46 fragment, originating from backbone cleavage at a m7G|U site, but cleavages at other G|U motifs were not enhanced. Based on the tRNA-Phe crystal structure, the m7G|U is found within the variable loop; however, significant secondary structure is not expected to be retained for tRNA-Phe after desalting and during CID. A more systematic exploration of fragmentation trends of a larger array of tRNA molecules is underway and may shed light on the production of preferential cleavages. Also observed in the CID spectrum of the tRNA-Phe isoform are several fragment ions with wellresolved isotopic distributions, as illustrated in Figure , that were not characterized by the automated fragment identification workflow. The detection of these additional unassigned fragment ions upon CID (as well as UVPD and a-EPD) suggests that additional neutral losses and/or secondary fragmentation may occur. Confirmation of the identities of these types of fragment ions is more challenging and requires greater statistical validation to ensure accurate assignment; this is an ongoing investigation.