Confident intact mass analysis of the tRNA isodecoders of phenylalanine by UHPLC-HRAM-MS and BioPharma Finder software

Significance of the Topic

In modern analytical chemistry confident detection and characterization of transfer RNA variants and their posttranscriptional modifications is essential for understanding translational fidelity and RNA function. Intact mass analysis offers rapid profiling with minimal sample preparation and high mass accuracy.

Objectives and Study Overview

This study demonstrates a workflow for intact mass analysis of phenylalanine transfer RNA isodecoders using ion pair reversed phase ultra high performance liquid chromatography coupled to high resolution accurate mass Orbitrap mass spectrometry and BioPharma Finder software. The aim was to achieve sensitive and accurate identification of tRNA variants and truncation species.

Methodology

• Ion pair reversed phase chromatography using a polymer based DNAPac RP column with dibutylamine and hexafluoroisopropanol mobile phase
• Gradient elution at 0.4 mL per minute on a biocompatible UHPLC platform maintained at 75 degrees Celsius
• Negative mode MS detection on an Orbitrap Ascend Tribrid instrument at 240000 resolution over 800 to 3000 m/z
• Data processing with BioPharma Finder intact mass workflow and Xtract deconvolution algorithm for monoisotopic mass extraction
• Creation of custom nucleobase modifications in software for accurate theoretical mass matching

Used Instrumentation

• Thermo Scientific Vanquish Horizon UHPLC system with binary pump and column compartment
• Thermo Scientific DNAPac RP column 2.1 by 100 mm 4 micron particle size
• Thermo Scientific Orbitrap Ascend Tribrid mass spectrometer
• Thermo Scientific BioPharma Finder software version 5.2
• Thermo Scientific Savant SpeedVac DNA 130 vacuum concentrator
• Thermo Scientific NanoDrop One micro volume spectrophotometer

Main Results and Discussion

The UHPLC HRAM MS analysis produced a clear charge state envelope from 9 negative to 26 negative with baseline resolved isotope patterns. Deconvolution revealed two major phenylalanine tRNA isodecoders with monoisotopic masses matching the A67 and G67 variants with mass deviations below 5 parts per million. Truncation series at the 3 prime terminus and a demethylated wybutosine species were also identified and quantified with relative abundance down to 3 percent.

Benefits and Practical Applications

• High confidence identification of intact tRNA variants and modifications with mass accuracy below 5 ppm
• Sensitive detection of low abundance species down to single digit percent relative abundance
• Fast and minimal sample preparation workflow without enzymatic digestion
• User friendly software for theoretical sequence editing data processing and annotation

Future Trends and Opportunities

Extending intact mass analysis to other regulatory RNA classes integration with top down fragmentation for localization of modifications and development of quantitative profiling methods in pharmaceutical quality control are promising directions. Advances in software automation and new ion source designs may further enhance sensitivity and throughput.

Conclusion

The combination of ion pair UHPLC and Orbitrap high resolution accurate mass spectrometry with BioPharma Finder software provides a robust platform for intact tRNA analysis. The approach yields confident identification of sequence variants and modifications with high mass accuracy and minimal sample preparation.

References

• Boccaletto P et al MODOMICS a Database of RNA Modification Pathways Nucleic Acids Research 2018
• Keith G and Dirheimer G Evidence for a Minor Variant tRNAPhe in Yeast Biochem Biophys Res Commun 1987
• Vanhinsbergh CJ et al Characterization of Large RNA and mRNA by Mass Spectrometry Anal Chem 2022
• Ross RL et al Characterization of mRNA 5 Capping Products Using LC HRAM MS BioPharma Finder Application Note 2022
• Kebarle P and Tang L Mechanism of Electrospray Mass Spectrometry Anal Chem 1993
• Noma A et al Biosynthesis of Wybutosine a Hypermodified Nucleoside in eukaryotic tRNA EMBO Journal 2006