MS1 and MS/MS spectra were recorded using either an Orbitrap Lumos mass spectrometer (Thermo Scientific Instruments) outfitted with a 193 nm excimer laser, enabling UVPD, or an Orbitrap Eclipse mass spectrometer (Thermo Scientific Instruments) for CAD acquisition. All MS1 and MS/MS spectra were acquired at 240,000 resolution during the elution period from 3 min to 8 min. A spray voltage of 1600 V was applied to a fused silica nanoelectrospray emitter (New Objective, 50 µm ID, 30 µm tip) via a stainless-steel tee and platinum electrode. MS/MS scans were acquired by performing quadrupole isolation of 2 m/z wide segments of the MS1 spectrum, with 0.5 m/z overlap of adjacent windows, prior to UVPD or CAD. The specific 2 m/z isolation width was selected to restrict the number of precursors per window, thereby limiting the heterogeneity in the MS/MS spectra. The 0.5 m/z overlap with adjacent windows was selected to ensure effective isolation of precursors at the edge of each isolation window. Windows spanned the range from m/z 859 to m/z 886, where the most abundant tRNA isoforms were observed, for a total of 25 windows. For UVPD, isolated precursor populations were irradiated with a single 0.75 mJ laser pulse applied during a 2 ms period. All UVPD was performed in the high-pressure linear ion trap. CAD spectra were acquired on an Orbitrap Eclipse mass spectrometer utilizing a normalized collision energy of 25 or 30 and a q value of 0.14, resulting in 50 total CAD spectra (corresponding to each of the twenty-five 2 m/z isolation/activation windows).