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( A ) Workflow for UPLC-MS/MS analysis of individual tRNAs. Separation of bulk tRNA by reversed-phase liquid chromatography (RP-LC) and 2D-PAGE of each fraction enabled isolation of all tRNA species. Cyanoethylation prior to electrophoresis revealed potential Ψ sites and allowed detection of s 4 U. Digestion with RNases T1, A, and U2 provided near-complete sequence coverage. Several deep-sequencing methods were applied to bulk tRNAs: RMS (RiboMethSeq), AAS (AlkAnilineSeq), GLORI (glyoxal/nitrite A deamination), HPS (HydraPsiSeq), BID (bisulfite-induced deletion). ( B to G ) Deconvoluted CID-MS/MS spectra of tRNA fragments containing modified nucleosides. The corresponding tRNA, RNase employed, and precursor ion (mass and charge) are indicated. Relevant y - and c -product ions are labeled. Insets show deep-sequencing signals. ( B ) [ce-s 4 U]8 in tRNA Trp (CCA), also detected by RT-signature. ( C ) [D]20 and [m 1 A]22 in tRNA Cys (GCA), confirmed by AAS and GLORI, respectively. ( D ) Modifications in the anticodon loop of tRNA Leu (UAA). RMS detected [Um]34, and Ψ (likely 39) was supported by HPS and BID. ( E ) [k 2 C]34 and [m 6 A]37 in tRNA Ile (CAU), also detected by AAS and GLORI, respectively. ( F ) [m 7 G]46 in tRNA Ala (UGC), validated by AAS. ( G ) [m 5 U]54 and [Ψ]55 in tRNA Ser (UGA). HPS detected both, while BID misassigned Ψ to -1 due to consecutive T54 and T55.

Journal: bioRxiv

Article Title: Complete post-transcriptional modification profiles in individual Staphylococcus aureus tRNA species

doi: 10.1101/2025.10.30.685614

Figure Lengend Snippet: ( A ) Workflow for UPLC-MS/MS analysis of individual tRNAs. Separation of bulk tRNA by reversed-phase liquid chromatography (RP-LC) and 2D-PAGE of each fraction enabled isolation of all tRNA species. Cyanoethylation prior to electrophoresis revealed potential Ψ sites and allowed detection of s 4 U. Digestion with RNases T1, A, and U2 provided near-complete sequence coverage. Several deep-sequencing methods were applied to bulk tRNAs: RMS (RiboMethSeq), AAS (AlkAnilineSeq), GLORI (glyoxal/nitrite A deamination), HPS (HydraPsiSeq), BID (bisulfite-induced deletion). ( B to G ) Deconvoluted CID-MS/MS spectra of tRNA fragments containing modified nucleosides. The corresponding tRNA, RNase employed, and precursor ion (mass and charge) are indicated. Relevant y - and c -product ions are labeled. Insets show deep-sequencing signals. ( B ) [ce-s 4 U]8 in tRNA Trp (CCA), also detected by RT-signature. ( C ) [D]20 and [m 1 A]22 in tRNA Cys (GCA), confirmed by AAS and GLORI, respectively. ( D ) Modifications in the anticodon loop of tRNA Leu (UAA). RMS detected [Um]34, and Ψ (likely 39) was supported by HPS and BID. ( E ) [k 2 C]34 and [m 6 A]37 in tRNA Ile (CAU), also detected by AAS and GLORI, respectively. ( F ) [m 7 G]46 in tRNA Ala (UGC), validated by AAS. ( G ) [m 5 U]54 and [Ψ]55 in tRNA Ser (UGA). HPS detected both, while BID misassigned Ψ to -1 due to consecutive T54 and T55.

Article Snippet: Electrophoresis was carried out in 1× TBE running buffer at 13 W for 24 h at 20°C using an S2 sequencing gel electrophoresis apparatus (BR Life Technologies).

Techniques: Tandem Mass Spectroscopy, Reversed-phase Chromatography, Isolation, Electrophoresis, Sequencing, Modification, Labeling