Biology subjects

Rusling, L.

Publications and source records attributed to Rusling, L..

4 recordsLinked to original sources

bioRxiv2026

Human 28S rRNA analysed by state-of-the-art oligonucleotide mass spectrometry: benchmarking current capabilities and a call to action for MS-Seq

Oligonucleotide mass spectrometry (MS-Seq) is emerging as a powerful approach for sequence-resolved RNA modification analysis, yet the field lacks standards for experimental workflows, data analysis and reporting. To assess current capabilities, the Human RNome Project Consortium conducted a cross-platform benchmarking study using a common RNA sample. A partial RNase T1 digest of human 28S rRNA was distributed to participating laboratories and analysed using existing LC-MS/MS workflows spanning different chromatographic strategies and mass spectrometers. To enable direct comparison, datasets were analysed using a harmonized NucleicAcidSearchEngine (NASE) workflow. Despite substantial methodological differences, laboratories recovered highly overlapping oligonucleotide sets and generated similar sequence coverage maps with a global coverage of 54.16%, demonstrating reproducible sequence information across platforms under standardized sample and analysis conditions. The benchmark further revealed incomplete sequence coverage, platform-specific differences in data architecture and increased assignment ambiguity during dynamic modification searches. Together with the community consensus developed during the HRPC workshop, these findings define priorities for the field, including improved sensitivity, standardized data analysis and reporting, community repositories, and robust bioinformatic workflows for confident de novo RNA modification discovery. This study provides an experimental benchmark and roadmap toward routine MS-based mapping of the human RNome. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/739151v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1fb1b9borg.highwire.dtl.DTLVardef@d172a5org.highwire.dtl.DTLVardef@bdc185org.highwire.dtl.DTLVardef@1ec1cc9_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Ion-Pair-Free Nanoflow HILIC-MS With RNase Benchmarking for Native RNA

RNA modifications play crucial roles in regulating cellular processes, but comprehensive mapping of the human RNome still remains limited by technological challenges. Mass spectrometry (MS) is a valuable tool to analyse RNA modifications complementing sequencing-based analysis. Current MS-based oligonucleotide workflows have limited sensitivity, requiring micrograms of RNA inputs and thus hindering studies on native RNAs. Additionally, environmentally toxic ion-pairing reagents are often required. Here, we report a highly sensitive, broadly applicable oligonucleotide-MS workflow that enables analysis of nanogram-scale RNA hydrolysates and we benchmark the substrate specificity of three nucleases: RNase T1, RNase 4, and colicin E5. We developed a nano-flow hydrophilic interaction liquid chromatography (HILIC) setup compatible with common MS buffers and coupled this with high-resolution MS. Using modified NucleicAcidSearchEngine (NASE), we confidently assigned RNA hydrolysates with diverse 3-end chemistries. Furthermore, we demonstrate that RNase 4 and colicin E5 efficiently cleave modified RNAs including pseudouridine-containing transcripts, enabling high sequence coverages. Using this workflow, we successfully mapped modifications in 25 ng of native yeast tRNAPhe and verified the sequence of 250 ng of a synthetic mRNA. Overall, our method provides a sensitive, high-resolution platform for oligonucleotide mass spectrometry, facilitating comprehensive analysis of RNA modifications and advancing efforts toward complete epitranscriptomic mapping. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=58 SRC="FIGDIR/small/689004v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@ed3dc3org.highwire.dtl.DTLVardef@171d3d6org.highwire.dtl.DTLVardef@a9ce2org.highwire.dtl.DTLVardef@c1d940_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Purity and stability of modified nucleosides in the context of accurate quantification by LC-MS

Accurate LC-MS (liquid chromatography coupled mass spectrometry) analysis of RNA modifications relies on synthetic nucleoside standards whose chemical integrity determines both qualitative identification and quantitative measurements. While the purity of these standards is typically verified prior to use, their long-term chemical stability during storage has not been systematically investigated. Here, we evaluated the stability of 44 canonical and modified ribonucleosides in aqueous solution during storage at -80 {degrees}C and -20 {degrees}C. Initial quality control confirmed the identity of all tested standards and revealed purity issues in selected compounds, including contamination of 1-methyladenosine (m1A) with 6-methyladenosine (m6A) and the presence of S- and R-isomers of 5-(carboxyhydroxymethyl)-2'-O-methyluridine (mchm5Um). Long-term LC-UV-MS monitoring over 12 months showed that 30 nucleosides remained stable, two were stable for at least six months, and 12 exhibited substantial quantitative changes. Seven nucleosides formed detectable degradation products, most of which could be structurally assigned. Quantum-chemical calculations of reaction free energies for deglycosylation, deamination, deacetylation and desulfurization correlated with the experimentally observed stability trends. Based on these results, we propose a practical guideline for the preparation, storage and analytical quality control of nucleoside standards, including recommendations for purity verification by UV spectroscopy and quantitative NMR. These guidelines provide an experimental framework to improve the robustness and inter-laboratory comparability of LC-MS-based RNA modification analysis.

molecular biology↗

bioRxiv2024

Vesicle-mediated mitochondrial clearance underlies an actionable metabolic vulnerability in triple-negative breast cancer

Selective autophagy of mitochondria is known to promote survival and progression of cancer cells in various malignancies including triple-negative breast cancer (TNBC). Here, we aimed to identify the essential metabolic adaptations that support mitochondrial quality control with the goal to uncover actionable metabolic vulnerabilities with therapeutic potential. Using an integrated approach of proteomics and untargeted and stable-isotope resolved metabolomics, coupled with functional experimental analyses, we define an alternative mechanism to mitophagy enabled by an onco-metabolic program of heightened extracellular sphingomyelin salvaging in TNBC that facilitates extracellular vesicle (EV)-mediated intracellular clearance of mitochondrial damage. Targeting of the cancer cell sphingolipid onco-metabolic pathway via repurposing of eliglustat, a selective small molecule inhibitor of glucosylceramide synthase (UGCG), resulted in ceramide-induced lethal mitophagy and attenuated tumor growth and prolonged overall survival at clinically achievable doses in an orthotopic syngeneic mouse model of TNBC. Our study defines a mechanism of aberrant sphingolipid metabolism that underlies an actionable metabolic vulnerability for anti-cancer treatment.

cancer biology↗