Biology subjects

Knittelfelder, O.

Publications and source records attributed to Knittelfelder, O..

8 recordsLinked to original sources

bioRxiv2026

Harmonized nucleoside mass spectrometry enables reproducible cross-platform RNA modification quantification

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 abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/739095v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@6e4e98org.highwire.dtl.DTLVardef@1ea13beorg.highwire.dtl.DTLVardef@b65858org.highwire.dtl.DTLVardef@1623337_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

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↗

bioRxiv2023

Lipidome unsaturation affects the morphology and proteome of the Drosophila eye.

While the proteome of an organism is largely determined by the genome, the lipidome is shaped by a poorly understood interplay of environmental factors and metabolic processes. To gain insights into the underlying mechanisms, we analyzed the impacts of dietary lipid manipulations on the ocular proteome of Drosophila melanogaster. We manipulated the lipidome with synthetic food media that differed in the supplementation of an equal amount of saturated or polyunsaturated triacylglycerols. This allowed us to generate flies whose eyes had a highly contrasting length and unsaturation of glycerophospholipids, the major lipid class of biological membranes, while the abundance of other membrane lipid classes remained unchanged. By bioinformatically comparing the resulting ocular proteomic trends and contrasting them with the impacts of vitamin A deficiency, we identified ocular proteins whose abundances are differentially affected by lipid saturation and unsaturation. For instance, we unexpectedly identified a group of proteins that have muscle-related functions and increase their abundances in the eye upon lipidome unsaturation but are unaffected by lipidome saturation. Moreover, we identified two differentially lipid-responsive proteins involved in stress responses, Turandot A and Smg5, whose abundances decrease with lipid unsaturation. Lastly, we discovered that the ocular lipid class composition is robust to dietary changes and propose that this may be a general homeostatic feature of the organization of eukaryotic tissues, while the length and unsaturation of fatty acid moieties is more variable to compensate environmental challenges. We anticipate that these insights into the molecular responses of the Drosophila eye proteome to specific lipid manipulations will guide the genetic dissection of the mechanisms that maintain visual function when the eye is exposed to dietary challenges.

neuroscience↗

bioRxiv2023

Eye proteome of Drosophila melanogaster

The Drosophila melanogaster eye is a popular model to elucidate the molecular mechanisms that underlie the structure and function of the eye as well as the causes of retinopathies. For instance, the Drosophila eye has been used to investigate the impacts of ageing and environmental stresses such as light-induced damage or dietary deficiencies. Moreover, large-scale screens have isolated genes whose mutation causes morphological and functional ocular defects, which includes key components of the phototransduction cascade. However, the proteome of the Drosophila eye is poorly characterized. Here, we used GeLC-MS/MS to quantify 3516 proteins he adult Drosophila melanogaster eye and provide a generic and expandable resource for further genetic, pharmacological, and dietary studies.

neuroscience↗

bioRxiv2020

Shotgun lipidomics and mass spectrometry imaging unveil diversity and dynamics in lipid composition in Gammarus fossarum

Sentinel species are playing an indispensable role in monitoring environmental pollution in aquatic ecosystems. Many pollutants found in water prove to be endocrine disrupting chemicals that could cause disruptions in lipid homeostasis in aquatic species. A comprehensive profiling of the lipidome of these species is thus an essential step towards understanding the mechanism of toxicity induced by pollutants. We here extensively examined both the composition and spatial distribution of lipids in freshwater crustacean Gammarus fossarum. The baseline lipidome of gammarids of different gender and reproductive stage was established by high throughput shotgun lipidomics. Spatial lipid mapping by high resolution mass spectrometry imaging led to the discovery of sulfate-based lipids in hepatopancreas and their accumulation in mature oocytes. We uncovered in G. fossarum a diverse and dynamic lipid composition that deepens our understanding of the biochemical changes during development and which could serve as a reference for future ecotoxicological studies.

biochemistry↗

bioRxiv2020

Alpha-synuclein enhances lipid droplet accumulation in neurons in a Drosophila model of Parkinson's disease

Parkinsons disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein (Syn) aggregation and associated with abnormalities in lipid metabolism. The accumulation of lipids in cytoplasmic organelles called lipid droplets (LDs) was observed in cellular models of PD. To investigate the pathophysiological consequences of interactions between Syn and proteins that regulate the homeostasis of LDs, we used a transgenic Drosophila model of PD, in which human Syn is specifically expressed in photoreceptor neurons. We first found that overexpression of the LD-coating proteins perilipin 1 or 2 (dPlin1/2), which limit the access of lipases to LDs, markedly increased triacylglyclerol (TG) loaded LDs in neurons. However, dPlin-induced-LDs in neurons are independent of lipid anabolic (diacylglycerol acyltransferase 1/Midway, fatty acid transport protein/dFatp) and catabolic (lipase Brummer) enzymes, indicating that non-canonical mechanisms regulate neuronal LD homeostasis. Interestingly, the accumulation of LDs induced by several distinct LD proteins (dPlin1, dPlin2, CG7900 or KlarsichtLD-BD) was synergistically amplified by the co-expression of Syn, which was found at the surface of LDs both in photoreceptors neurons of Drosophila and in human neuroblastoma cells. Finally, the accumulation of LDs increased the resistance of Syn to proteolytic digestion, a phenomenon associated with Syn aggregation in human neurons. We thus propose that Syn cooperates with LD proteins to inhibit lipolysis and that binding of Syn to LDs contributes to the pathogenic misfolding and aggregation of Syn in neurons.

cell biology↗

bioRxiv2019

Sterols as dietary markers for Drosophila melanogaster

During cold acclimation fruit flies switch their feeding from yeast to plant food, however there are no robust markers to monitor it in the wild. Drosophila melanogaster is a sterol auxotroph and relies on dietary sterols to produce lipid membranes, lipoproteins and molting hormones. We employed shotgun lipidomics to quantify eight major food sterols in total extracts of heads, female and male genital tracts of adult flies. We found that their sterol composition is dynamic and reflective of flies diet in an organ-specific manner. Season-dependent changes observed in the organs of wild-living flies suggested that the molar ratio between yeast (ergosterol, zymosterol) and plant (sitosterol, stigmasterol) sterols is a quantifiable, generic and unequivocal marker of their feeding behavior, including cold acclimation. It provides technically simpler and more contrast readout compared to the full lipidome analysis and is suitable for ecological and environmental population-based studies.

biochemistry↗

bioRxiv2018

Body size-dependent energy storage causes Kleibers law scaling of the metabolic rate in planarians

Kleibers law, or the [3/4]-power law scaling of the metabolic rate with body mass, is considered one of the few quantitative laws in biology, yet its physiological basis remains unknown. Here, we report Kleibers law scaling in the planarian Schmidtea mediterranea. Its reversible and life history-independent changes in adult body size over 2 orders of magnitude reveal that Kleibers law does not emerge from the size-dependent decrease in cellular metabolic rate, but from a size-dependent increase in mass per cell. Through a combination of experiment and theoretical analysis of the organismal energy balance, we further show that the mass allometry is caused by body size dependent energy storage. Our results reveal the physiological origins of Kleibers law in planarians and thus have general implications for understanding a fundamental scaling law in biology.

biophysics↗