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Structural Localization of Mass-Degenerate Intact tRNAPHE Species by Ion-Pair UHPLC-HRMS and CID-MS3
Robert L Ross1, Hangyan Sun2, Markus Bursch3
1Thermo Fisher Scientific, Division of Chromatography and Mass Spectrometry, Lexington, Massachusetts 02421, United States.
Abstract:
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-MS3 structural localization to resolve mass-degenerate species in yeast tRNAPHE. Intact profiling of wild-type (WT) and ΔTYW3 strains revealed a reproducible -14 Da subpopulation that was indistinguishable at the neutral-mass level. CID-MS3 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.
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