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Mass spectrometry-based Quantitative RNA Modification Mapping Using Isobaric Tandem Mass Tags

ABSTRACT: Here, we present an RNA-specific isobaric tandem mass tagging (RMT) approach that enables the quantitative mapping of RNA modifications at an omics scale with high chemical accuracy. These RNA mass tags, derived and optimized from proteomics isobaric tags, are incorporated into an end-to-end workflow spanning sample preparation, LC-MS/MS acquisition, and customized data analysis. Validation using synthetic RNA oligonucleotides and total tRNAs from Pseudomonas aeruginosa UCBPP-PA14 outstanding quantification reproducibility, with coefficients of variation consistently below 5%. Applying this pipeline, we identified and quantified 22 RNA modifications in PA14 tRNAs, including two novel tRNA modification sites-m2A at position 38 and Gm/Cm at position 39-and their corresponding writer enzymes. Further analyses of tRNAs from writer enzyme knockout strains and cells cultured under stress conditions revealed dynamic modification patterns, interdependencies among modification pathways, novel insights into writer enzyme function and their potential roles in cellular stress adaptation. Our method provides a robust, and cost-effective platform for quantitative RNA modification mapping, enabling deeper biological insights and advancing RNA-related biomedical research.

INSTRUMENT(S):

ORGANISM(S): Pseudomonas Aeruginosa (strain Ucbpp-pa14)

SUBMITTER: Junzhou Wu

LAB HEAD: Peter Dedon

PROVIDER: PXD066919 | Pride | 2026-07-01

REPOSITORIES: Pride

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			Action	DRS
	69mer_iTRAQ_R1.idXML	Other
	69mer_iTRAQ_R1.raw	Raw
	69mer_iTRAQ_R2.idXML	Other
	69mer_iTRAQ_R2.raw	Raw
	69mer_iTRAQ_R3.idXML	Other

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