Highlights

• A novel challenge is addressed in the study, focusing on the separation and detection difficulties of highly polar oligonucleotides, which typically lack retention on standard reversed phase chromatographic columns and exhibit poor signal intensity in mass spectrometry analysis.
• The study introduces an innovative ion-pair free HPLC-Linear Trap Quadrupole (LTQ)/Orbitrap-Mass spectrometry (MS) method for tRNA modification profiling. This method involves selective, simple, and rapid pre-column derivatization of MTBSTFA, enhancing the efficiency and specificity of tRNA analysis.
• A groundbreaking finding is presented, indicating the upregulation of U[m1G][m2G] in nonalcoholic fatty liver disease (NAFLD) cells, suggesting a previously unreported link between tRNA modification and NAFLD. This discovery adds a novel dimension to our understanding of tRNA modification’s potential role in disease.

Summary

A novel labeling method utilizing N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide (MTBSTFA) has been developed for nucleic acid analysis. This approach offers significant advantages, including strong retention, easily interpretable data, and the ability to detect modified bases.

Researchers at the Macau University of Science and Technology, led by Huixia Zhang (2023), have successfully applied this method to profile tRNA, demonstrating its potential in characterizing tRNAs in nonalcoholic fatty liver disease (NAFLD).

The study also highlights the development of a pre-column derivatization method using MTBSTFA for tRNA modification profiling. This method enables the analysis of RNA modifications and has proven effective in both in vitro and in vivo studies. It suggests a connection between tRNA modification, specifically U[m1G][m2G] upregulation, and NAFLD. Mass spectrometry, ion-pairing reagents, and hydrophilic interaction liquid chromatography are valuable tools for nucleic acid characterization and enhanced retention and detection of oligonucleotides. The study’s findings, including significant changes in tRNA fragments and the upregulation of a unique tRNAAsn(QUU) digestion product in NAFLD cells, are accessible along with the analysis data and code at https://pubs.acs.org/doi/10.1021/acs.analchem.2c02302.

T.-M. Yan, Y. Pan, M.-L. Yu, K. Hu, K.-Y. Cao, and Z.-H. Jiang, “Full-Range Profiling of tRNA Modifications Using LC–MS/MS at Single-Base Resolution through a Site-Specific Cleavage Strategy,” Anal. Chem., vol. 93, no. 3, pp. 1423–1432, Jan. 2021, doi: .