Quantitative LC-MS Analysis of tRNA Modifications

Total RNA was isolated using Trizol RNA extraction. Small RNAs were subsequently purified from total RNA using the Zymo RNA Clean & Concentrator-5 kit. Small RNAs (1 μg) was digested and analyzed by LC-MS as previously described (Dewe et al., 2017 (link); Zhang et al., 2020 (link); Cai et al., 2015 (link)). Briefly, ribonucleosides were separated using a Hypersil GOLD C18 Selectivity Column (Thermo Scientific) followed by nucleoside analysis using a Q Exactive Plus Hybrid Quadrupole-Orbitrap. The modification difference ratio was calculated using the m/z intensity values of each modified nucleoside following normalization to the sum of intensity values for the canonical ribonucleosides; A, U, G, and C. Statistical analysis of the mass spectrometry results was performed using GraphPad Prism software with error bars representing the standard deviation. Statistical tests and the number of times each experiment was repeated are noted in the figure legend. Raw intensity values for each measured nucleoside are provided in the source data file.

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Zhang K., Eldin P., Ciesla J.H., Briant L., Lentini J.M., Ramos J., Cobb J., Munger J, & Fu D. (2024). Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease. eLife, 12, RP90316.

Publication 2024

RNA Clean & Concentrator-5 kit Hypersil GOLD C18 Selectivity Column Q Exactive Plus Hybrid Quadrupole-Orbitrap

Corresponding Organization :

Other organizations : University of Rochester, Institut de Recherche en Infectiologie de Montpellier, Université de Montpellier, Centre National de la Recherche Scientifique, University of Rochester Medical Center

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Variable analysis

independent variables

Total RNA isolation using Trizol RNA extraction
Small RNA purification using Zymo RNA Clean & Concentrator-5 kit

dependent variables

Modifications in small RNAs analyzed by LC-MS

control variables

Nucleoside analysis using a Q Exactive Plus Hybrid Quadrupole-Orbitrap
Ribonucleosides separation using a Hypersil GOLD C18 Selectivity Column (Thermo Scientific)
Normalization of m/z intensity values to the sum of intensity values for the canonical ribonucleosides; A, U, G, and C

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

Annotations

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