Serial dilutions were prepared using commercial pure ribonucleosides (0.005-150 pg, Carbosynth, Toronto Research Chemicals) in order to establish the linear range of quantification and the limit of detection of each compound. A mix of commercial ribonucleosides was injected before and after each batch of samples to assess instrument stability and to be used as an external standard to calibrate the retention time of each ribonucleoside.
"],"repository":["MetaboLights"],"study_status":["Public"],"ptm_modification":[""],"instrument_platform":["Liquid Chromatography MS - positive - reverse phase"],"chromatography_protocol":["Samples were analyzed using an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled to an EASY-nLC 1200 (Thermo Fisher Scientific (Proxeon), Odense, Denmark). Ribonucleosides were loaded directly onto the analytical column and were separated by reversed-phase chromatography using a 50-cm column with an inner diameter of 75 μm, packed with 2 μm C18 particles (Thermo Fisher Scientific, cat ES903). Chromatographic gradients started at 93% buffer A and 3% buffer B with a flow rate of 250 nl/min for 5 minutes and gradually increased to 30% buffer B and 70% buffer A in 20 min. After each analysis, the column was washed for 10min with 0% buffer A and 100% buffer B. Buffer A: 0.1% formic acid in water. Buffer B: 0.1% formic acid in 80% acetonitrile.
"],"publication":["Selective profiling of translationally active tRNAs and their dynamics under stress."],"submitter_name":["Mie Monti"],"submitter_affiliation":["CRG"],"organism_part":["MCF-7"],"technology_type":["mass spectrometry assay"],"extraction_protocol":["Ribosomes for the ribo-Embedded tRNAs were isolated using the RiboLace kit (Immagina Biotechnology S.r.l., cat. no: GF001-12), following manufacturer’s recommendations. RNA was then eluted from ribosomes using the following RNA Clean & ConcentratorTM-5 kit (Zymo catalog. No. R1015 or R1016) according to manufacturer instructions, with a modification: 200 uL of Zymo RNA Binding Buffer was added directly to the beads, followed by thorough mixing. Recovered RNA was quantified at 260 nm using a Nanodrop ND-1000 UV-VIS Spectrophotometer, and RNA integrity and size were assessed via 15% TBE-UREA gels and/or Agilent 4200 TapeStation RNA HS ScreenTape Assay (cat. no: 5067-5579). 100 ng of small RNA (<200 nt) fraction, separated as described above, were digested with 1 μl of the Nucleoside Digestion Mix (New England BioLabs, M0649S) and the mixture was incubated at 37 ºC for 1 h.
Acquired data were analyzed with the Skyline-daily software (v24.1.1.254) and extracted precursor areas of the ribonucleosides were used for quantification.
For methionine deprivation, MCF-7 cells at ~70% confluency were washed with pre-warmed PBS and incubated in methionine-free medium (MFM) for 6–16 hours. MFM was prepared using methionine-free DMEM (Thermo Fisher, cat. no: 21013024), supplemented with 10% dialyzed FBS (Gibco, cat. no: A3382001) and 1% (v/v) penicillin–streptomycin (Thermo Fisher, cat. no: 15140122). Control medium was generated by supplementing MFM with L-methionine to a final concentration of 0.2 mM (Thermo Fisher, cat. no: J6190418). All experimental and control conditions were prepared in parallel and maintained under identical incubation parameters.
The mass spectrometer was operated in positive ionization mode with nanospray voltage set at 2.4 kV and source temperature at 275°C. For the Parallel Reaction Monitoring (PRM) method, the quadrupole isolation window was set to 1.4 m/z, and MS/MS scans were collected over a mass range of m/z 50-450, with detection in the Orbitrap at the resolution of 60,000. MSMS fragmentation of defined masses with schedule retention time was performed using HCD at NCE 20, the auto gain control (AGC) was set to “Standard” and a maximum injection time of 118 ms was used. In each PRM cycle, one full MS scan at a resolution of 120,000 was acquired over a mass range of m/z 220-700 with detection in the Orbitrap mass analyzer. Auto gain control (AGC) was set to 1e5 and the maximum injection time was set to 50 ms.
"],"metabolite_name":["5-methyluridine","inosine","1-methylinosine","queuosine","2'O-methyladenosine","Guanosine","m6Am","2'-O-Methyl-5-methyluridine","5-hydroxymethylcytidine","5-Carbamoylmethyluridine","2-methyladenosine","8-methyladenosine","3-(3-Amino-3-carboxypropyl)uridine","7-methylguanosine","N6-threonylcarbamoyladenosine","uridine","dihydrouridine","3-methylpseudouridine","3-methyluridine","1-methylpseudouridine","6-methyladenosine","Adenosine","2-Methylguanosine","1-methyladenosine","N2,N2-Dimethylguanosine","cytidine","2'-O-methylguanosine","5-methoxycarbonylmethyluridine","pseudouridine","3-methylcytidine","8-Oxoguanosine","5-methylcytidine","5-Formylcytidine","5-Carboxymethyluridine","6-hydroxymethyladenosine","1-Methylguanosine","5-Methoxycarbonylmethyl-2-thiouridine"],"additional_accession":[]},"is_claimable":false,"name":"Selective profiling of translationally active tRNAs and their dynamics under stress","description":"During translation, transfer RNAs (tRNAs) deliver specific amino acids to the ribosome in a coordinated manner with the sequence encoded by the mRNA. Despite their central role in protein synthesis, the precise contribution of tRNAs to the modulation of translation remains poorly understood, primarily due to lack of methods to characterise tRNA abundances and their modifications from actively translating ribosomes. Here we develop RiboNano-tRNAseq, a novel method to quantify native ribosome-associated tRNAs (ribo-tRNAs) from actively translating ribosomes, achieved by coupling tag-free pulldown of active ribosomes with native tRNA nanopore sequencing. Applying RiboNano-tRNAseq, we investigate how the ribosome-associated tRNAome varies under diverse stress conditions. Our work reveals highly tailored tRNA responses depending on the stress type. For example, under leucine starvation, we observe enrichment of a subset of tRNALeu isoacceptors in ribo-tRNAs. By contrast, methionine starvation leads to a decrease in methyl-based tRNA modifications, such as m1A or m2,2G, reflecting the decreased availability of the methyl donor S-adenosylmethionine (SAM). Strikingly, arsenite starvation does not affect tRNA modification profiles, but instead causes a drastic fragmentation of tRNAs, predominantly within ribosome-associated tRNAs. Altogether, our work findings demonstrate that RiboNano-tRNAseq enables comprehensive characterization of the tRNAome and its dynamics by simultaneously capturing tRNA abundance, modification and fragmentation patterns, both in total and actively translating tRNA pools.
","dates":{"publication":"2026-06-25","submission":"2025-08-01"},"accession":"MTBLS12806","cross_references":{"MetaboLights":["MTBLC17562","MTBLC17802","MTBLC23774","MTBLC16704","MTBLC20129","MTBLC20607","MTBLC19068","MTBLC178091","MTBLC45996","MTBLC89487","MTBLC16335","MTBLC17596","MTBLC234279","MTBLC19226","MTBLC191041","MTBLC16020","MTBLC19688","MTBLC69426","MTBLC178092","MTBLC21891","MTBLC19065","MTBLC16750","MTBLC229469","MTBLC20794","MTBLC19062","MTBLC19229","MTBLC19702","MTBLC229468","MTBLC40304","MTBLC62005","MTBLC75654","MTBLC19289","MTBLC20598","MTBLC20597","MTBLC19928","MTBLC60193","MTBLC21440"],"ChEBI":["CHEBI:17562","CHEBI:17802","CHEBI:23774","CHEBI:16704","CHEBI:20129","CHEBI:20607","CHEBI:19068","CHEBI:178091","CHEBI:45996","CHEBI:89487","CHEBI:16335","CHEBI:17596","CHEBI:234279","CHEBI:19226","CHEBI:191041","CHEBI:16020","CHEBI:19688","CHEBI:69426","CHEBI:178092","CHEBI:21891","CHEBI:19065","CHEBI:16750","CHEBI:229469","CHEBI:20794","CHEBI:19062","CHEBI:19229","CHEBI:19702","CHEBI:229468","CHEBI:40304","CHEBI:62005","CHEBI:75654","CHEBI:19289","CHEBI:20598","CHEBI:20597","CHEBI:19928","CHEBI:60193","CHEBI:21440"]}}