Robert L. Ross

RNA modification analysis by LC–MS/MS is central to epitranscriptomics, yet quantitative comparison across laboratories and instrument platforms remains poorly standardized. Here, we performed a community-driven benchmarking study during the first Human RNome Project workshop to systematically evaluate cross-platform reproducibility of ribonucleoside mass spectrometry workflows. Using the same analytical column and gradient, standardized RNA samples, and shared reagents, we compared nucleoside quantification across quadrupole, time-of-flight, and orbitrap-based LC–MS platforms employing distinct acquisition strategies. While chromatographic separation was highly reproducible across systems, nucleoside-specific MS response behavior differed substantially between platforms and limited direct comparability of relative signal intensities. These response differences varied across analytes and concentration ranges, demonstrating that harmonized chromatography alone is insufficient for transferable quantitative analysis. Stable isotope-labeled internal standard (SILIS) normalization substantially reduced platform-and method-dependent response and improved agreement for most evaluated modifications. External calibration improved agreement between qTOF and Orbitrap workflows for a subset of modifications but did not fully resolve residual intersystem differences. Based on these findings, we establish benchmark-derived recommendations for harmonized relative and absolute RNA modification quantification, including guidance for calibration design, quality control, and data reporting. Together, this work provides a methodological framework for reproducible nucleoside LC–MS/MS workflows and establishes a foundation for large-scale comparative epitranscriptomic studies. Graphical

Intact mass spectrometry of structured RNAs preserves full-length molecular context but is intrinsically susceptible to mass degeneracy arising from single-methyl variations and biosynthetic intermediates. Neutral mass measurements alone are insufficient for unambiguous structural assignment. Here, we demonstrate a structured analytical strategy integrating ion-pair UHPLC-high-resolution mass spectrometry (IP-UHPLC-HRMS), nucleoside-level HCD confirmation, and CID-MS³ structural localization to resolve mass-degenerate species in yeast tRNA^PHE. Intact profiling of wild-type (WT) and ΔTYW3 strains revealed a reproducible -14 Da subpopulation that was indistinguishable at the neutral-mass level. CID-MS³ of the base-loss fragment established backbone identity, while UVPD preserved a precursor m/z shift consistent with a 14 Da neutral mass difference localized to the wybutosine (yW) modification. These orthogonal measurements localized the -14 Da difference without reliance on database-only assignments or fragment-level inference. The approach is illustrated using the yW pathway as a defined test case for resolving intact RNA mass degeneracy.

The RNA-binding protein La is found in most eukaryotes, and despite being essential in many organisms, its function is not completely clear. Trypanosoma brucei, the causative agent of human African trypanosomiasis, encodes a ‘classical’ La protein (TbLa) composed of a La-motif, two RNA recognition motifs (RRM1 and RRM2α), a C-terminal short basic motif (SBM), and a nuclear localization signal (NLS). In T. brucei, like in most eukaryotes, position 34 of tRNATyr, -Asp, -Asn and -His is modified with queuosine (Q34). The steady-state levels of queuosine-modified tRNA in the insect form (procyclic) of T. brucei can fluctuate dynamically depending on growth conditions, but the mechanism(s) controlling Q34 levels are not well understood. A well-established function of La is in precursor-tRNA 3′-end metabolism, but in this work, we demonstrate that La also controls Q34-tRNA levels. Individual domain deletions showed that while deletion of La motif or RRM1 causes dysregulation of Q34-tRNA levels, no other domain plays a similar role. We also show that La is important for the normal balance of several additional tRNA modifications. These findings are discussed in the context of substrate competition between La and modification enzymes, also highlighting subcellular localization as a key determinant of tRNA function.

Nucleic acid oligonucleotides represent a promising therapy for treating disease. Production of these biopolymers happens synthetically which can produce impurities concomitant with the desired product. The use of liquid chromatography coupled to high resolution mass spectrometry is a robust analytical technique for determination of sample purity. Traditionally, ion pairing reagents are used to facilitate chromatography separation before sampling with the mass spectrometer. The choice of reagent for analysis can impact the analysis as the length of the oligonucleotide increases. This is in part due to the thermodynamics of transitioning the oligonucleotide from the liquid phase to the gas phase. Here we show that the use of a hydrogen bond donating alkylamine assists with overcoming the energy barrier of phase transition by bonding to the nucleobase as well as the phosphodiester backbone. By increasing the relative hydrophobicity of the oligonucleotide, the transition to the gas phase is increased producing a cleaner mass spectrum. Furthermore, we show that by using a hydrogen bond donating alkylamine, we can remove the fluoroalcohol reagent commonly used in ion pairing mass spectrometry of nucleic acids.

Transfer ribonucleic acid (tRNA) are the smallest RNA in the translational triune and contain the greatest density of post-transcriptional modifications than any other RNA types in the cell. Due to the size of tRNA studying these modifications usually entails enzymatic digestion followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Here we report an advancement in Intact Mass Analysis for identification of tRNA through deconvolution of high resolution accurate mass spectrometry facilitated using a secondary alkylamine as an ion pairing reagent during reverse phase chromatography. We identify in isolated and total S. cerevisiae tRNA 3’ CCA variations and show that most tRNA lack a 3’ adenosine with the lesser abundant species having the expected CCA termini. We identify a previously unknown stable demethylated Wybutosine intermediate for tRNA ^PHE and identify low abundant contaminating tRNAs in an isolated tRNA ^PHE sample. Confirmation of identities was verified through traditional mass spectrometric nucleoside and mass mapping experiments.

Extensive knowledge of both the nature and position of tRNA modifications in all cellular tRNAs has been limited to two bacteria, Escherichia coli and Mycoplasma capricolum. Bacillus subtilis sp subtilis strain 168 is the model Gram-positive bacteria and the list of the genes involved in tRNA modifications in this organism is far from complete. Mass spectrometry analysis of bulk tRNA extracted from B. subtilis, combined with next generation sequencing technologies and comparative genomic analyses, led to the identification of 41 tRNA modification genes with associated confidence scores. Many differences were found in this model Gram-positive bacteria when compared to E. coli. In general, B. subtilis tRNAs are less modified than those in E. coli, even if some modifications, such as m¹A22 or ms²t⁶A, are only found in the model Gram-positive bacteria. Many examples of non-orthologous displacements and of variations in the most complex pathways are described. Paralog issues make uncertain direct annotation transfer from E. coli to B. subtilis based on homology only without further experimental validation. This difficulty was shown with the identification of the B. subtilis enzyme that introduces ψ at positions 31/32 of the tRNAs. This work presents the most up to date list of tRNA modification genes in B. subtilis, identifies the gaps in knowledge, and lays the foundation for further work to decipher the physiological role of tRNA modifications in this important model organism and other bacteria.

Streptomyces ghanaensis ATCC14672 is remarkable for its production of phosphoglycolipid compounds, moenomycins, which serve as a blueprint for the development of a novel class of antibiotics based on inhibition of peptidoglycan glycosyltransferases. Here we employed mariner transposon (Tn) mutagenesis to find new regulatory genes essential for moenomycin production. We generated a library of 3000 mutants which were screened for altered antibiotic activity. Our focus centred on a single mutant, HIM5, which accumulated lower amounts of moenomycin and was impaired in morphogenesis as compared to the parental strain. HIM5 carried the Tn insertion within gene ssfg_01967 for putative tRNA (N6-isopentenyl adenosine(37)-C2)-methylthiotransferase, or MiaB, and led to a reduced level of thiomethylation at position 37 in the anticodon of S. ghanaensis transfer ribonucleic acid (tRNA). It is likely that the mutant phenotype of HIM5 stems from the way in which ssfg_01967::Tn influences translation of the rare leucine codon UUA in several genes for moenomycin production and life cycle progression in S. ghanaensis . This is the first report showing that quantitative changes in tRNA modification status in Streptomyces have physiological consequences.

Post-transcriptional chemical modifications of (t)RNA molecules are crucial in fundamental biological processes, such as translation. Despite their biological importance and accumulating evidence linking them to various human diseases, technical challenges have limited their detection and accurate quantification. Here, we present a sensitive capillary nanoflow liquid chromatography mass spectrometry (nLC-MS) pipeline for quantitative high-resolution analysis of ribonucleoside modifications from complex biological samples. We evaluated two porous graphitic carbon (PGC) materials and one end-capped C18 reference material as stationary phases for reversed-phase separation. We found that these matrices have complementing retention and separation characteristics, including the capability to separate structural isomers. PGC and C18 matrices yielded excellent signal-to-noise ratios in nLC-MS while differing in the separation capability and sensitivity for various nucleosides. This emphasizes the need for tailored LC-MS setups for optimally detecting as many nucleoside modifications as possible. Detection ranges spanning up to six orders of magnitude enable the analysis of individual ribonucleosides down to femtomol concentrations. Furthermore, normalizing the obtained signal intensities to a stable isotope labeled spike-in enabled direct comparison of ribonucleoside levels between different samples. In conclusion, capillary columns coupled to nLC-MS constitute a powerful and sensitive tool for quantitative analysis of modified ribonucleosides in complex biological samples. This setup will be invaluable for further unraveling the intriguing and multifaceted biological roles of RNA modifications.