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Updated: Jul 18, 2026

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications
Published on: July 10, 2020
A novel method for sequence placement of modified nucleotides in mixtures of transfer RNA
Tracy M Wagner1, Vinod Nair, Rebecca Guymon
1Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
Abstract:
Sequence placement of post-transcriptionally modified nucleosides in tRNA can be experimentally difficult, particularly in cases involving new or unexpected modifications or sequence sites. We describe a mass spectrometry-based approach to this problem, involving the following steps: crude isolations of one or several tRNAs by HPLC from an unfractionated tRNA mixture; digestion to oligonucleotide mixtures by RNase T1; analysis by combined HPLC/electrospray ionization-MS for recognition of modifications; and direct gas-phase sequencing of selected targets in the mixture by LC/MS/MS. Isoacceptor identity can be established in favorable cases when tRNA gene sequences are available.
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