# Mass Spectrometry of Modified RNAs

> **NIH NIH R01** · UNIVERSITY OF CINCINNATI · 2024 · $345,835

## Abstract

PROJECT SUMMARY
Transfer ribonucleic acids (tRNAs) are required for the synthesis of proteins within cells. tRNAs
are found to contain the highest density and greatest diversity of post-transcriptionally modified
nucleosides of any class of RNAs. Our scientific understanding of the functional role of these
modified nucleosides in tRNAs is limited in large part by the lack of methods that can routinely
identify and map modifications onto a primary tRNA sequence. The long-term goal of this research
continues to be to develop appropriate mass spectrometric approaches that enable biological
studies into the functional significance of modified tRNAs. This renewal is focused on the specific
goal of identifying and understanding modified tRNAs from human samples. This research is
timely and relevant as less than 15% of the nearly 270 predicted unique human tRNA sequences
have been characterized at this level of detail. Moreover, a number of recent studies have
implicated tRNAs and their modifications in a variety of different human diseases and pathologies.
Our knowledge of the complete modification profiles of human tRNAs has been limited by the lack
of bioanalytical methods capable of identifying specific modified ribonucleosides and particular
sequence locations within individual tRNAs. Based on our advances in the last support period,
the three specific aims in this renewal address the short-comings in the field by establishing a
workflow that will define the population of tRNAs expressed in the sample, the census of modified
nucleosides present, and the mapping of those modified nucleosides onto the specific expressed
tRNAs. Our innovations will lead to the creation of tRNA region-specific modification profiling
assays that can be used to track dynamic changes in these modifications across multiple sample
types. We will demonstrate the utility and significance of our approach by examining changes in
these profiles using melanocytes and various melanomas.
This research plan will have significant impacts on multiple fields and disciplines. When complete,
we will have an approach that can document the qualitative and quantitative differences of
modified tRNA expressed in tissue, cell lines or disease condition. Our advances will provide the
ability to develop insights into the role of tRNA modification status in protein synthesis regulation,
proteostasis imbalances or disease state maintenance. Moreover, the bioanalytical developments
to result from this research can be applied to scientific investigations that seek to understand how
the cell regulates RNA modification patterns as well as how variable RNA modification patterns
affect other cellular regulatory processes.

## Key facts

- **NIH application ID:** 10746428
- **Project number:** 5R01GM058843-19
- **Recipient organization:** UNIVERSITY OF CINCINNATI
- **Principal Investigator:** PATRICK A LIMBACH
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $345,835
- **Award type:** 5
- **Project period:** 1999-02-01 → 2025-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10746428

## Citation

> US National Institutes of Health, RePORTER application 10746428, Mass Spectrometry of Modified RNAs (5R01GM058843-19). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10746428. Licensed CC0.

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