Structural Localization of Mass-Degenerate Intact tRNA ^PHE Species by Ion-Pair UHPLC–HRMS and CID–MS ³

Intact mass spectrometry of structured RNAs preserves full-length molecular context but is intrinsically susceptible to mass degeneracy arising from single-methyl variations and biosynthetic intermediates. Neutral mass measurements alone are insufficient for unambiguous structural assignment. Here, we demonstrate a structured analytical strategy integrating ion-pair UHPLC–high-resolution mass spectrometry (IP-UHPLC–HRMS), nucleoside-level HCD confirmation, and CID–MS 3 structural localization to resolve mass-degenerate species in yeast tRNA PHE . Intact profiling of wild-type (WT) and ΔTYW3 strains revealed a reproducible −14 Da subpopulation that was indistinguishable at the neutral-mass level. CID–MS 3 of the base-loss fragment established backbone identity, while UVPD preserved a precursor m / z shift consistent with a 14 Da neutral mass difference localized to the wybutosine (yW) modification. These orthogonal measurements localized the -14 Da difference without reliance on database-only assignments or fragment-level inference. The approach is illustrated using the yW pathway as a defined test case for resolving intact RNA mass degeneracy.