Proteomic mapping of novel tubulin post-translational modifications in Trypanosoma cruzi cytoskeleton Microtubules (MTs) play central roles in the organization and morphology of trypanosomatid parasites, forming highly specialized cytoskeletal structures such as the subpellicular corset, the flagellar axoneme, and the mitotic spindle. Functional specialization of MTs is regulated by the "tubulin code", which is defined by the combination of different - and {beta}-tubulin isotypes, a set of post-translational modifications (PTMs) and specific MT-binding proteins. Although multiple tubulin PTMs have been described in trypanosomatids using specific antibodies or mass spectrometry, to date no comprehensive mapping has been reported in Trypanosoma cruzi, the causative agent of Chagas Disease. In the present work, we performed a high-resolution proteomic analysis of PTMs present in - and {beta}-tubulin subunits of the T. cruzi Dm28c strain, using tubulin-enriched extracts obtained by in vitro polymerization. Multiple PTMs were identified, including acetylation, methylation, phosphorylation, and polyglutamylation, for which many modified amino acids had not been previously reported in trypanosomatids. Structural mapping of these modifications onto a predicted /{beta}-tubulin heterodimer showed that most modified residues are located in solvent-exposed regions of the protein. Together, these findings provide the first systematic map of tubulin PTMs in T. cruzi and support the existence of a complex tubulin code contributing to microtubule regulation in this parasite.

(BioRxiv All) Proteomic mapping of novel tubulin post-translational modifications in Trypanosoma cruzi cytoskeleton: Microtubules (MTs) play central roles in the organization and morphology of trypanosomatid parasites, forming highly specialized cytoskeletal structures such as… #BioRxiv #MassSpecRSS

Molecular dissection of protein complexes isolated from sections of human brain Molecular studies of brain receptors and transporters have typically relied on recombinant systems, limiting insight into their organization in native tissue. Here, we develop nanobody-based immunoprecipitation coupled with native mass spectrometry to interrogate endogenous protein assemblies from post-mortem mouse and human brain sections. We exemplify our approach by characterizing the synaptic proteins VGluT1 and mGluR2. From a single mouse brain, we discover mGluR2/3 heterodimers, alongside mGluR2 homodimers. Considering regions of human brain heterodimeric mGluR2/3 is highly abundant in the OFC and sgACC (~70% and 50%, respectively) and forms regional-specific interactions with additional synaptic proteins. In a modest cohort of biobanked human tissue, associated with depression and suicide, we find increased mGluR2/3 in the OFC. Consistent with this, mice exhibit similar associations between heterodimer levels and stress-susceptibility. Overall, our approach provides a direct means for establishing molecular-behavioural links at the level of receptor organization in brain.

(BioRxiv All) Molecular dissection of protein complexes isolated from sections of human brain: Molecular studies of brain receptors and transporters have typically relied on recombinant systems, limiting insight into their organization in native tissue. Here, we develop… #BioRxiv #MassSpecRSS

Enzymatic Ligation Strategy to Enhance Electrospray Ionization Efficiency and Liquid Chromatography-Mass Spectrometry of DNA and RNA Oligonucleotides Mass spectrometry (MS) is a powerful technique for characterizing modified RNA as it directly sequences and quantifies all mass-altering modifications simultaneously. However, the physicochemical properties of RNA result in poor ionization efficiencies during electrospray ionization, presenting a major barrier to sensitive MS measurements necessary for low abundance RNA samples and RNAs with low modification stoichiometries. Here, we report a ligation-based approach to increase ionization efficiencies of RNA oligonucleotides. We show that short (~5 nt), chemically modified DNA oligonucleotides can be enzymatically ligated to RNA to serve as MS signal enhancers. Among a series of signal enhancers appended with various alkyl and alkylimidazolium functional groups, we found that decyl-functionalized derivatives improved MS sensitivity by ~15-fold compared to unlabeled oligonucleotide. When ligated to RNA standards, the decyl-modified signal enhancer increased MS signals 2-4-fold with the additional benefit of improved retention during liquid chromatography (LC) separations without ion pairing agents. To apply the ligation-based approach to RNase T1 digests of longer RNAs, a multi-step enzymatic approach was optimized to maximize ligation efficiencies. We then ligated signal enhancers to a yeast transfer RNA (tRNA) digest and observed increased MS signals for numerous sequence-informative digestion products. Importantly, the sequences of RNA oligonucleotides ligated to signal enhancers were readily determined by tandem mass spectrometry with collision-induced dissociation. This ligation-based strategy for enhancing LC-MS/MS characterization of RNA creates opportunities to measure low abundance RNA samples and their modifications.

(BioRxiv All) Enzymatic Ligation Strategy to Enhance Electrospray Ionization Efficiency and Liquid Chromatography-Mass Spectrometry of DNA and RNA Oligonucleotides: Mass spectrometry (MS) is a powerful technique for characterizing modified RNA as it directly sequences and… #BioRxiv #MassSpecRSS

A Joint Promoterome-Proteome Atlas Highlights the Molecular Diversity of Human Skeletal Muscles More than 600 distinct skeletal muscles constitute up to 40% of the total mass of the human body. Human skeletal muscles differ in anatomical position, morphology, origin, and function, but the diversity of their molecular phenotypes, the gene expression and protein abundance profiles, remains poorly explored. Here, we report the large-scale CAGE-Seq promoterome profiling of 75 human skeletal muscles, complemented by 22 matched proteomes obtained with mass spectrometry. We identified 37001 transcribed regulatory elements and 1804 protein groups encompassing 1895 proteins, 80% of which demonstrated non-uniform expression across different muscles. The skeletal muscles of the eye, tongue, and diaphragm had the most distinctive molecular phenotypes, while the overall diversity was driven by hundreds of transcription factors with tissue-specific activity. By analyzing the allelic imbalance of CAGE-Seq reads, we discovered 6653 allele-specific single-nucleotide variants often coinciding with muscle-related GWAS SNPs, including muscle volume. Finally, we provide an interactive online atlas of transcriptomic and proteomic molecular phenotypes, facilitating further studies of gene regulation and heritable pathologies of skeletal muscles.

(BioRxiv All) A Joint Promoterome-Proteome Atlas Highlights the Molecular Diversity of Human Skeletal Muscles: More than 600 distinct skeletal muscles constitute up to 40% of the total mass of the human body. Human skeletal muscles differ in anatomical position, morphology,… #BioRxiv #MassSpecRSS

Stage-dependent tau post-translational modifications map the spatiotemporal progression of Alzheimer's disease Alzheimer's disease (AD) is defined by progressive tau aggregation, yet the molecular events driving this process remain poorly understood. Post-translational modifications (PTMs) are key regulators of tau biology and potential biomarkers of disease progression. Using immunoprecipitation-mass spectrometry and absolute quantification of tau isoforms, we profiled tau PTMs in soluble and insoluble brain fractions. We studied multiple brain regions (hippocampus, inferior temporal and frontal gyri), representing regions affected at different stages of pathology, from human donors spanning the AD spectrum and staged by ABC neuropathological scoring. We uncovered a stage-dependent PTM landscape across AD progression: early phosphorylation changes, including pT217 and pS262, precede later ubiquitination events, such as uK311, associated with tau aggregation. We also identified PTMs negatively correlated with aggregation, including mK258, suggesting potential protective roles. These findings refine our understanding of the spatiotemporal evolution of tau biochemistry and offer mechanistic and translational insights into AD tauopathy.

(BioRxiv All) Stage-dependent tau post-translational modifications map the spatiotemporal progression of Alzheimer's disease: Alzheimer's disease (AD) is defined by progressive tau aggregation, yet the molecular events driving this process remain poorly understood.… #BioRxiv #MassSpecRSS

🚨 New lab preprint alert! 🚨
A study 6 years in the making, made possible by @simonsfoundation.org support : "A multi-omic atlas in the African turquoise killifish reveals increased glucocorticoid signaling as a hallmark of brain aging" now on #biorXiv: www.biorxiv.org/content/10.6... A 🧵 1/11 🧪🖥️🧬

Statistical Principles Define an Open-Source Differential Analysis Workflow for Mass Spectrometry Imaging Experiments with Complex Designs Mass spectrometry imaging (MSI) characterizes the spatial heterogeneity of molecular abundances in biological samples. Experiments with complex designs, involving multiple conditions and multiple samples, provide particularly useful insight into differential abundance of analytes. However, analyses of these experiments require attention to details such as signal processing, selection of regions of interest, and statistical methodology. This manuscript contributes a statistical analysis workflow for detecting differentially abundant analytes in MSI experiments with complex designs. Using a case study of histologic samples of human tibial plateaus from knees of osteoarthritis patients and cadaveric controls, as well as simulated datasets, we illustrate the impact of the analysis decisions. We illustrate the importance of signal processing and feature aggregation for preserving biological relevance and alleviating the stringency of multiple testing. We further demonstrate the importance of selecting regions of interest in ways that are compatible with differential analysis. Finally, we contrast several common statistical models for differential analysis, showcase the appropriate use of replication, and demonstrate model-based calculation of sample size for followup investigations. The discussion is accompanied by detailed recommendations and an open-source R-based implementation that can be followed by other investigations.

(BioRxiv All) Statistical Principles Define an Open-Source Differential Analysis Workflow for Mass Spectrometry Imaging Experiments with Complex Designs: Mass spectrometry imaging (MSI) characterizes the spatial heterogeneity of molecular abundances in biological samples.… #BioRxiv #MassSpecRSS

Deep learning enables direct HLA typing from immunopeptidomics data The immune system eliminates malignant and infected cells through T-cell-mediated recognition of peptides presented by human leukocyte antigen molecules. Mass spectrometry-based immunopeptidomics enables unbiased identification of naturally presented HLA-restricted peptides and has become central to the development of T-cell-based immunotherapies. However, immunopeptidomics data reflects the combined peptide presentation of multiple HLA alleles, and determining which allotypes are represented in this multi-allelic complexity remains an unmet computational challenge. Here, we introduce immunotype, a deep learning-based ensemble predictor for HLA class I allotyping directly from immunopeptidomics data. Immunotype integrates peptide and HLA sequence information through transformer encoders and a graph neural network, complemented by a curated mono-allelic reference of known peptide-HLA binding preferences. Immunotype achieves an overall accuracy of 87.2% at protein-level resolution across diverse tissues and thereby enables rapid, cost-effective HLA typing of large-scale immunopeptidomics datasets.

(BioRxiv All) Deep learning enables direct HLA typing from immunopeptidomics data: The immune system eliminates malignant and infected cells through T-cell-mediated recognition of peptides presented by human leukocyte antigen molecules. Mass spectrometry-based… #BioRxiv #MassSpecRSS

Critical amino acid residues in the N-terminal domain of NADPH-dependent assimilatory sulfite reductase flavoprotein mediate octameric assembly How large, flexible enzymes assemble into defined oligomeric architectures remains a central question in biology. NADPH-dependent assimilatory sulfite reductase (SiR) forms a heterododecamer built on an octameric flavoprotein (SiRFP) core, yet the molecular basis for this assembly has been unresolved because of its disordered N-terminus. Here, we use ion mobility mass spectrometry, small-angle neutron scattering, and mutagenesis to define the mechanism of SiRFP oligomerization. We show that SiRFP forms a discrete, stable octamer in solution. We also report that its N-terminal 52-residue segment is necessary and sufficient to mediate assembly, also mediating oligomerization when fused to a heterologous protein. Structure-guided mutagenesis identifies four residues (Gln22, Tyr39, Phe40, and Gln47) whose substitution disrupts the octamer, producing concentration-dependent lower-order species while retaining catalytic activity. These findings define the determinants of SiRFP assembly with broader implications for engineering homomeric protein complexes.

(BioRxiv All) Critical amino acid residues in the N-terminal domain of NADPH-dependent assimilatory sulfite reductase flavoprotein mediate octameric assembly: How large, flexible enzymes assemble into defined oligomeric architectures remains a central question in biology.… #BioRxiv #MassSpecRSS

Comparison of Extraction Methods for the Quantification of Phytohormones from Tomato Fruits and Leaves by LC-MS/MS Accurate, simultaneous, and efficient quantification of chemically diverse phytohormone species is a critical task towards understanding the complex system of phytohormone signaling pathways. Quantification of phytohormones with the commonly used technique liquid chromatography coupled to tandem mass spectrometry is susceptible to the influence of non-phytohormone components present in the sample, a phenomenon referred to as matrix effect. To reduce matrix effect, some phytohormone quantification methods include additional steps of cleanup of crude extracts. However, to what extent additional purification steps provide increased accuracy compared to simpler, less laborious methods is seldomly evaluated. We evaluated three previously described phytohormone extraction methods, two of which include solid-phase extraction and one that does not, in their ability to minimize matrix effect and generate accurate estimates of phytohormone species spanning six classifications, from fruit and leaf tissue of Solanum lycopersicum cv. Micro-Tom (tomato). Our results show that, while the methods that included solid phase extraction occasionally outperformed each other regarding matrix effect and/or recovery efficiency for broad range of phytohormones, they rarely outperformed the simpler single-phase extraction method.

(BioRxiv All) Comparison of Extraction Methods for the Quantification of Phytohormones from Tomato Fruits and Leaves by LC-MS/MS: Accurate, simultaneous, and efficient quantification of chemically diverse phytohormone species is a critical task towards understanding the… #BioRxiv #MassSpecRSS

Soil bacterium Massilia secretes metabolites that promote Leptospira growth Understanding pathogen metabolism is critical for identifying key functions for drug targeting, establishing effective in vitro experimental systems, etc., particularly for metabolically unique organisms such as Leptospira. Pathogenic Leptospira are thought to infect humans from environmental sources; however, direct isolation from environmental samples remains technically challenging and is not yet well established. Here, we report that a ubiquitous environmental bacterium, Massilia sp., produces metabolites that promote the growth of Leptospira interrogans, encountered through an incidental contamination event, and identified in this study. Gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis showed demonstrated that cultivating of Massilia sp. in R2A medium resulted in the accumulation of metabolites, including branched-chain amino acid (BCAA) intermediates, compared to fresh medium. By combining genome-scale metabolic modeling with experimental validation using cell-free culture supernatant supplementation assays, we demonstrate that BCAA intermediates, particularly 2-ketoisocaproic acid (4-methyl-2-oxopentanoate; 4MOP), a leucine biosynthetic intermediate produced by Massilia sp., enhance Leptospira growth. To investigate the metabolic role of 4MOP, we incorporated transcriptomic data into a genome-scale metabolic network model to generate condition-specific models. Resulted flux distributions indicated that Leptospiracatabolized imported 4MOP to produce acetyl-CoA. Our results reveal a previously unrecognized metabolic interaction where metabolites produced by environmental bacteria support the growth of pathogenic Leptospira, offering mechanistic insight into its metabolic requirement. These findings have implications to understand the environmental persistence of Leptospira through its metabolic dependencies on coexisting microbes, and they also help develop better strategies for this pathogen.

(BioRxiv All) Soil bacterium Massilia secretes metabolites that promote Leptospira growth: Understanding pathogen metabolism is critical for identifying key functions for drug targeting, establishing effective in vitro experimental systems, etc., particularly for metabolically… #BioRxiv #MassSpecRSS

Defining the RNA Modification Landscape of Multiple Myeloma Reveals METTL3-Dependent m6A Regulation of NEAT1 RNA modifications play critical roles in gene regulation. N6-methyladenosine (m6A) is the most abundant modification on mRNA and long noncoding RNA (lncRNA) and regulates RNA processing, stability, and translation. RNA modifications are not well characterized in multiple myeloma (MM), a plasma cell malignancy characterized by relapse and disease progression, and the contribution of m6A -modified lncRNAs to the disease remains unclear. Here, we define the RNA modification landscape of MM by combining mass spectrometry, Nanopore Direct RNA sequencing, and methylated RNA immunoprecipitation sequencing. We identify 20 RNA modification types and > 15,000 m6A sites, including sites on 2,398 lncRNAs. Among these, we validate m6A sites on the paraspeckle-associated lncRNA NEAT1. Functional studies reveal that NEAT1 expression is regulated by the methyltransferase METTL3 and site-specific demethylation of a NEAT1 m6A site reduces MM cell viability. Single-cell RNA sequencing shows consistent NEAT1 enrichment in malignant plasma cells but minimal expression in healthy cells. These findings identify m6A-modified lncRNAs as key regulators of MM biology and establish NEAT1 as an epitranscriptomically controlled driver of MM cell survival.

(BioRxiv All) Defining the RNA Modification Landscape of Multiple Myeloma Reveals METTL3-Dependent m6A Regulation of NEAT1: RNA modifications play critical roles in gene regulation. N6-methyladenosine (m6A) is the most abundant modification on mRNA and long noncoding RNA… #BioRxiv #MassSpecRSS

Defining the RNA Modification Landscape of Multiple Myeloma Reveals METTL3-Dependent m6A Regulation of NEAT1 RNA modifications play critical roles in gene regulation. N6-methyladenosine (m6A) is the most abundant modification on mRNA and long noncoding RNA (lncRNA) and regulates RNA processing, stability, and translation. RNA modifications are not well characterized in multiple myeloma (MM), a plasma cell malignancy characterized by relapse and disease progression, and the contribution of m6A -modified lncRNAs to the disease remains unclear. Here, we define the RNA modification landscape of MM by combining mass spectrometry, Nanopore Direct RNA sequencing, and methylated RNA immunoprecipitation sequencing. We identify 20 RNA modification types and > 15,000 m6A sites, including sites on 2,398 lncRNAs. Among these, we validate m6A sites on the paraspeckle-associated lncRNA NEAT1. Functional studies reveal that NEAT1 expression is regulated by the methyltransferase METTL3 and site-specific demethylation of a NEAT1 m6A site reduces MM cell viability. Single-cell RNA sequencing shows consistent NEAT1 enrichment in malignant plasma cells but minimal expression in healthy cells. These findings identify m6A-modified lncRNAs as key regulators of MM biology and establish NEAT1 as an epitranscriptomically controlled driver of MM cell survival.

(BioRxiv All) Defining the RNA Modification Landscape of Multiple Myeloma Reveals METTL3-Dependent m6A Regulation of NEAT1: RNA modifications play critical roles in gene regulation. N6-methyladenosine (m6A) is the most abundant modification on mRNA and long noncoding RNA… #BioRxiv #MassSpecRSS

Defining the RNA Modification Landscape of Multiple Myeloma Reveals METTL3-Dependent m6A Regulation of NEAT1 RNA modifications play critical roles in gene regulation. N6-methyladenosine (m6A) is the most abundant modification on mRNA and long noncoding RNA (lncRNA) and regulates RNA processing, stability, and translation. RNA modifications are not well characterized in multiple myeloma (MM), a plasma cell malignancy characterized by relapse and disease progression, and the contribution of m6A -modified lncRNAs to the disease remains unclear. Here, we define the RNA modification landscape of MM by combining mass spectrometry, Nanopore Direct RNA sequencing, and methylated RNA immunoprecipitation sequencing. We identify 20 RNA modification types and > 15,000 m6A sites, including sites on 2,398 lncRNAs. Among these, we validate m6A sites on the paraspeckle-associated lncRNA NEAT1. Functional studies reveal that NEAT1 expression is regulated by the methyltransferase METTL3 and site-specific demethylation of a NEAT1 m6A site reduces MM cell viability. Single-cell RNA sequencing shows consistent NEAT1 enrichment in malignant plasma cells but minimal expression in healthy cells. These findings identify m6A-modified lncRNAs as key regulators of MM biology and establish NEAT1 as an epitranscriptomically controlled driver of MM cell survival.

(BioRxiv All) Defining the RNA Modification Landscape of Multiple Myeloma Reveals METTL3-Dependent m6A Regulation of NEAT1: RNA modifications play critical roles in gene regulation. N6-methyladenosine (m6A) is the most abundant modification on mRNA and long noncoding RNA… #BioRxiv #MassSpecRSS

A Context-Aware Single-Cell Proteomics Analysis pipeline. Single-cell proteomics (SCP) by mass spectrometry can now quantify hundreds to thousands of proteins per cell, but the field still lacks standardised analytical pipelines that accommodate the diversity of instruments, sample preparation workflows and biological contexts encountered in practice. Existing workflows, largely adapted from single-cell transcriptomics, do not account for the informative missingness, pervasive ambient protein contamination and limited feature space that distinguish proteomic from transcriptomic data. In addition, cell type annotation remains a manual bottleneck that is subjective, difficult to reproduce and hard to scale. Here we present an end-to-end pipeline that integrates adaptive quality control, entropy-guided iterative batch correction, multi-modal marker discovery that exploits detection patterns unique to proteomics, and context-aware annotation by large language models (LLMs) coupled to structured contradiction reasoning and orthogonal data-driven validation. Benchmarking on published single-cell proteomic datasets from developing human brain and glioblastoma-associated neutrophils revealed systematic LLM failure modes, including context-insensitive marker vocabulary and misinterpretation of phagocytic or lytic cell states. We addressed these errors using a three-round prompt architecture that combines general biological principles with auto-generated dataset-specific constraints. In held-out validation on a skin tumour dataset acquired, the pipeline showed high concordance with FACS-sorted ground truth. In the caerulein-injured pancreas, orthogonal immunohistochemistry further supported annotations of macrophage, stellate and immune populations. The pipeline is fully automated under fixed settings, and available as Context-Aware Single-Cell Proteomics Analysis (CASPA), providing SCP laboratories and facilities with a reproducible workflow that delivers interpretable, confidence-quantified annotations suitable for downstream expert review.

(BioRxiv All) A Context-Aware Single-Cell Proteomics Analysis pipeline.: Single-cell proteomics (SCP) by mass spectrometry can now quantify hundreds to thousands of proteins per cell, but the field still lacks standardised analytical pipelines that accommodate the diversity of… #BioRxiv #MassSpecRSS

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Molecular architecture of the ciliary base in mammalian multiciliated cells Multiciliated epithelial cells (MCCs) generate tens to hundreds of motile cilia to drive fluid flow in diverse physiological contexts. While the axonemal structure of motile cilia has been described extensively in recent years, the molecular architecture of the transition zone, basal body, and surrounding ciliary environment of MCCs remain more elusive. Here, we use cryo-focused ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET) to obtain in situ 3D views of the ciliary base within intact MCCs from mammalian trachea, complemented by in situ cross-linking mass spectrometry (XL/MS) and ultrastructure expansion microscopy (U-ExM) for molecular identification. Our data reveal spatially-defined modifications of microtubule architecture from the proximal centriole to the early axoneme, including transition zone-specific features such as an A-B linker bridging microtubule doublets and a helical assembly of microtubule inner proteins (MIPs). We show that the ciliary necklace, a feature observed in many motile cilia, is spatially aligned with the transition zone and quantify its regular organization within the membrane. Our in situ data capture rarely observed events, including intraflagellar transport (IFT) trains connecting to ciliary vesicles tethered to undocked centrioles. The surrounding ciliary environment contains intermediate filaments that encircle the basal bodies and bundled actin filaments that elaborate microvilli structures between the cilia. Integration of XL/MS and U-ExM identified novel microtubule associated proteins (MAPs), MIPs, and membrane-associated proteins localized to these distinct subdomains. This work provides a molecular and structural map of the mammalian MCC ciliary base, revealing architectural principles that underlie its assembly, organization, and function.

(BioRxiv All) Molecular architecture of the ciliary base in mammalian multiciliated cells: Multiciliated epithelial cells (MCCs) generate tens to hundreds of motile cilia to drive fluid flow in diverse physiological contexts. While the axonemal structure of motile cilia has… #BioRxiv #MassSpecRSS

NovoTax: prokaryotic strain identification from mass spectrometry-based proteomics data Traditional mass spectrometry-based proteomics typically requires prior knowledge of sample composition to match spectra to peptides. Yet, novel de novo peptide sequencing approaches can provide peptide sequences to identify the organism. Here, we introduce an end-to-end pipeline (NovoTax) to identify the closest prokaryotic genome directly from raw bottom-up proteomics data. The approach combines peptide sequencing tools with an optimized implementation of peptide searching through an extensive genome database. On a benchmark dataset of species isolates, we identified the reported species and strain in the majority of the cases, and showed that in discordant cases NovoTax was likely correct. Interestingly, NovoTax was also able to identify contaminating species in some samples. The algorithm also identified the most abundant species in bacterial communities. In summary, NovoTax provides strain level identification of microbial samples enabling the downstream use of traditional proteomics search engines for a deeper proteome analysis.

(BioRxiv All) NovoTax: prokaryotic strain identification from mass spectrometry-based proteomics data: Traditional mass spectrometry-based proteomics typically requires prior knowledge of sample composition to match spectra to peptides. Yet, novel de novo peptide sequencing… #BioRxiv #MassSpecRSS

Hepatitis B virus proteome analysis identifies apolipoprotein C1 facilitating particle production and virus entry Background & Aims: Antiviral therapies targeting hepatitis B virus (HBV) suppress viral replication, but rarely achieve functional cure. Understanding HBV-host cell interaction is crucial for developing novel therapeutic approaches. Here, we report host cell proteins associated with HBV virions and filamentous subviral particles (fSVPs) and characterize one of them, apolipoprotein C1 (ApoC1), mechanistically. Methods: Highly purified HBV virions and fSVPs were obtained by sequential use of several biophysical methods. Particles were analyzed by mass spectrometry and associated proteins were evaluated phenotypically using an HBV infection model. The top hit, ApoC1 was characterized in detail. Results: Associated with virions and fSVPs, we identified in addition to known chaperones such as HSP90AB1 and HSC70, several apolipoprotein-related factors. RNAi-based phenotypic validation identified strongest effects for ApoC1, likely due to two complementary effects. First, ApoC1 depletion reduced intracellular cholesterol level impairing HBV infection and SVP production, which was compensated by exogenous cholesterol substitution. Second, ApoC1 that is mainly enriched in high-density lipoprotein (HDL), associates with HBV virions and fSVPs and increases HBV infectivity. The same was found for hepatitis D virus (HDV), a satellite virus utilizing HBV envelopes. Supplementation of exogenous HDL enhanced infection most likely via scavenger receptor class B type 1 (SR-B1), the natural HDL receptor. Consistently, inhibition of SR-B1 suppressed HBV and HDV infection. Conclusions: We established a method for obtaining highly purified HBV virions and fSVPs and identified the HDL component ApoC1 to associate with both particle types. ApoC1 promotes HBV and HDV infection most likely via SR-B1 facilitating viral entry.

(BioRxiv All) Hepatitis B virus proteome analysis identifies apolipoprotein C1 facilitating particle production and virus entry: Background & Aims: Antiviral therapies targeting hepatitis B virus (HBV) suppress viral replication, but rarely achieve functional cure.… #BioRxiv #MassSpecRSS

The long noncoding RNA Dory is required for female but not male spatial learning and memory Large numbers of long noncoding RNAs (lncRNAs) exhibit region- or cell-specific expression and subcellular locations in the mammalian brain. We analyzed the expression and function of the mouse lncRNA 2700046G09Rik (also called Sgms1os1), which we have named Dory due to sex-specific disruption of spatial memory in rodent gene knockout and RNA knockdown. We show that Dory is predominantly expressed in a punctate pattern in nuclei of excitatory neurons in the hippocampus, and in both neuronal and non-neuronal cells in the cerebellum. We show by high resolution RNA sequencing that Dory brain transcripts are composed of 1-3 exons with multiple isoforms. Deletion of the constitutive first exon of Dory by CRISPR-Cas9 genome editing resulted in the impairment of spatial memory and some aspects of balance in female but not male mice. Dory-null mice presented without overt changes in brain morphology. Knockdown of the rat homolog of Dory in dorsal hippocampus using antisense oligonucleotides confirmed inhibition of spatial memory in females only. RNA sequencing and mass spectrometry revealed differential hippocampal gene and protein expression profiles, notably of prolactin, growth hormone and pro-opiomelanocortin, between male and female Dory knockout mice.

(BioRxiv All) The long noncoding RNA Dory is required for female but not male spatial learning and memory: Large numbers of long noncoding RNAs (lncRNAs) exhibit region- or cell-specific expression and subcellular locations in the mammalian brain. We analyzed the expression… #BioRxiv #MassSpecRSS

Selonsertib-Eluting Electrode Coating Attenuates Cochlear Injury Pathways Cochlear implants partially restore sound sensation for individuals with severe to profound hearing loss. This can significantly improve the recipients quality of life, largely through improved communication with spoken language. However, surgical trauma from electrode insertion also instigates biological responses that can compromise device performance and longevity. To address these limitations, we designed a novel polymer based coating that provides controlled release of selonsertib, an apoptosis signal regulating kinase 1 (ASK1) inhibitor with well described anti inflammatory, anti-fibrosis and cytoprotective properties. We describe the physical and molecular evaluation of this coating, including the application of next generation mass spectrometry based proteomics in mammalian cochlear explants and their culture medium. These analyses provide new insights into the acute tissue response in the damaged cochlea and demonstrate that selonsertib eluted from the polymer coating has a significant pro survival, anti inflammatory, and anti fibrosis effect. This study establishes proof of concept for selonsertib eluting polymers, demonstrating both feasibility for targeted drug delivery and efficacy for mitigating acute cochlear damage responses.

(BioRxiv All) Selonsertib-Eluting Electrode Coating Attenuates Cochlear Injury Pathways: Cochlear implants partially restore sound sensation for individuals with severe to profound hearing loss. This can significantly improve the recipients quality of life, largely through… #BioRxiv #MassSpecRSS

Mitochondrial and Cardiolipin Adaptations to Ventricular Assist Device Support in Pediatric Versus Adult Failing Myocardium Background: Ventricular assist devices (VADs) are used as treatment for end-stage heart failure in children and adults. We previously demonstrated decreased mitochondrial function and changes in cardiolipin, a mitochondrial phospholipid, in explanted pediatric and adult failing hearts. In this study, we tested the hypothesis that VAD unloading of failing hearts leads to positive changes in myocardial cardiolipin in both pediatric and adult hearts. Methods: Ventricular tissue was collected from the same patient at time of VAD implantation and at transplant. Ejection fraction (EF), left ventricular internal diameter at end-diastole (LVIDd) and brain natriuretic peptide (BNP) were assessed pre- and post-VAD. Cardiolipin species from paired VAD core and explants were quantified using liquid chromatography mass spectrometry. Mitochondrial respiration was measured in ventricular tissue pre- and post-VAD in paired pediatric samples using the Oroboros Oxygraph-2k. Results: VAD support led to increased EF and decreased LVIDd and BNP. The predominant cardiolipin species in cardiac mitochondria, tetralinoleoylcardiolipin, was positively remodeled in pediatric post-VAD myocardium, while adult post-VAD myocardium demonstrated significantly increased total cardiolipin and decreased oxidized cardiolipin but did not demonstrate the tetralinoleoylcardiolipin remodeling seen in pediatric hearts. In pediatric patients, VAD support resulted in significant increases in Complex I+II activity, and a trend toward increases in Complex I activity. Conclusion: Our data demonstrate age-related differences in VAD-associated cardiolipin remodeling and suggest that improved mitochondrial function in pediatric VAD-supported hearts could be related to increased tetralinoleoylcardiolipin.

(BioRxiv All) Mitochondrial and Cardiolipin Adaptations to Ventricular Assist Device Support in Pediatric Versus Adult Failing Myocardium: Background: Ventricular assist devices (VADs) are used as treatment for end-stage heart failure in children and adults. We previously… #BioRxiv #MassSpecRSS

Gallium induces cytotoxicity through disruption of DNA synthesis rather than ferroptosis Background: Gallium (Ga) is a promising anti-tumor agent; however, its precise molecular targets in osteosarcoma remain debated. While current paradigms largely attribute its toxicity to reactive oxygen species (ROS) and ferroptosis, understanding its true mechanism is essential for overcoming therapeutic resistance. This highlights the need for interdisciplinary approaches, such as metabolomics, to unveil novel vulnerabilities in cancer metabolism. Methods:,We employed an interdisciplinary strategy utilizing high-resolution liquid chromatography-mass spectrometry (LC-MS) metabolomics and 13C2-glutamine stable isotope tracing in osteosarcoma cells to elucidate the cytotoxic mechanisms of gallium nitrate. Scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDS) was utilized for elemental mapping, and in silico modeling was applied to evaluated metal binding dynamics. Furthermore, synergistic effects were tested by combining gallium with the DNA-damaging agent cisplatin. Results: Our metabolic profiling revealed a profound bifurcation characterized by the systemic depletion of glycolysis and pentose phosphate pathway intermediates, coupled with a novel ribonucleotide accumulation bottleneck. The observed distinct signature strongly implicated ribonucleotide reductase (RNR) as the primary enzymatic target. In silico modeling and SEM-EDS visually and thermodynamically confirmedthat gallium acts as a structural decoy for iron within the RNR active site. The co-localization induces functional iron starvation rather than canonical ferroptosis. Furthermore, isotope tracing confirmed that elevated ROS is a consequence of overall metabolic failure, not the primary driver of cell death. Crucially, gallium functioned as a metabolic DNA repair inhibitor, synergizing potently with cisplatin to prevent the repair of platinum-induced DNA lesions. Conclusions: Gallium selectively sensitizes highly proliferative sarcoma cells by disrupting RNR-mediated DNA precursor synthesis, while sparing normal osteoblasts. Leveraging metabolomics to uncover this state of functional iron starvation provides a rational, interdisciplinary framework for developing gallium-based combination therapies designed to break platinum resistance in clinical oncology.

(BioRxiv All) Gallium induces cytotoxicity through disruption of DNA synthesis rather than ferroptosis: Background: Gallium (Ga) is a promising anti-tumor agent; however, its precise molecular targets in osteosarcoma remain debated. While current paradigms largely attribute… #BioRxiv #MassSpecRSS

Proteomic profiling of whole tissue sections in cardiac ATTR amyloidosis reveals increased extracellular matrix remodeling Cardiac transthyretin amyloidosis (ATTR-CA) is caused by myocardial deposition of misfolded transthyretin, leading to progressive heart failure. Disease pathology, however, extends beyond passive amyloid deposition and also involves active processes such as extracellular matrix (ECM) remodeling and immune activation. Mass spectrometry (MS) is the gold standard for amyloid typing in diagnostics. Here, we applied quantitative MS-driven proteomics on formalin-fixed paraffin-embedded whole cardiac tissue sections from six ATTR-CA cases, ten unaffected controls and four AL-CA controls to investigate protein expression changes. In addition to transthyretin, over 500 proteins were upregulated in ATTR-CA biopsies, including complement and coagulation factors as well as extracellular matrix (ECM) remodeling proteins. Among these, members of the A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) family, metalloproteinases (MMPs), and Tissue Inhibitor of Metalloproteinases (TIMP3) showed significant upregulation. These proteins are key regulators of ECM turnover and structural integrity. Immunohistochemistry confirmed ADAMTS4 enrichment in amyloid deposits, while TIMP3 showed strong expression in cardiomyocytes and weaker staining within amyloid deposits. Together, these findings indicate that ECM remodeling, alongside complement and coagulation activation, represents a reproducible feature of cardiac ATTR amyloidosis. Whole-tissue proteomics provides biological insights that extend beyond amyloid typing, with potential implications for biomarker discovery and therapeutic targeting in ATTR-CA.

(BioRxiv All) Proteomic profiling of whole tissue sections in cardiac ATTR amyloidosis reveals increased extracellular matrix remodeling: Cardiac transthyretin amyloidosis (ATTR-CA) is caused by myocardial deposition of misfolded transthyretin, leading to progressive heart… #BioRxiv #MassSpecRSS

Proteome analyses reveal Endoplasmic Reticulum stress-induced changes in protein abundance associated with Ube2j2 deficiency in human cell culture The unfolded protein response (UPR) helps reinstate cellular proteostasis upon an accumulation of misfolded proteins in the endoplasmic reticulum (ER), in part through ER-associated degradation (ERAD). Ube2j2 is an ER-localized E2 ubiquitin-conjugating enzyme that participates in ERAD. We used mass spectrometry analysis of cultured U2OS cells to investigate how the loss of Ube2j2 affects the cellular proteome in response to tunicamycin-induced ER stress. We constructed a network of twelve statistically distinct modules of protein abundance profiles across conditions. We describe the Gene Ontology annotations for each module along with the hub gene proteins whose abundance levels most closely adhere to each modules protein abundance profile. Our analysis identifies known Ube2j2-associated pathways (e.g., the UPR and ERAD) and cellular functions that were previously unassociated with Ube2j2 (e.g., RNA metabolism, ER-Golgi transport, and cell-cycle progression). These data are available via ProteomeXchange with identifier PXD076153 and provide avenues for further investigation into the cellular functions of Ube2j2 under basal and ER-stressed conditions.

(BioRxiv All) Proteome analyses reveal Endoplasmic Reticulum stress-induced changes in protein abundance associated with Ube2j2 deficiency in human cell culture: The unfolded protein response (UPR) helps reinstate cellular proteostasis upon an accumulation of misfolded… #BioRxiv #MassSpecRSS

A multi-omics approach to identify the impact of miR-411ed on NSCLC TKI resistance Tyrosine Kinase inhibitors (TKIs) are widely used as effective chemotherapeutic agents for treating patients with EGFR-mutated NSCLC. Unfortunately, after treatment, patients eventually develop resistance to TKI therapy. The most common resistance mechanism for the TKI Osimertinib is the overexpression of the MET Proto-Oncogene, Receptor Tyrosine Kinase (MET). We previously demonstrated that miR-411-5p A-to-I edited at position 5 (miR-411ed) can directly target MET in A549 and H1299 cells. MiR-411ed in combination with Osimertinib reduced cell proliferation in two TKI resistant EGFR-mutated cell lines: HCC827R and PC9R. MiR-411ed did not downregulate MET expression in HCC827R, suggesting an alternative mechanism for TKI response. In this study, we aim to identify the mechanism of miR-411ed TKI response using a multi-omics approach of RNAseq and protein mass spectrometry. In our cellular model, we identified miR-411ed affected genes independent of MET activity, resulting in 211 genes (RNAseq) and 36 proteins (proteomics). Pathway analysis identified an increase in interferon signaling for RNAseq and combined omics, and a decrease in ERK/MAPK signaling in proteomics. Using the IsoTar target prediction tool, we identified STAT3 as a key regulator and confirmed STAT3 protein downregulation upon transfection with miR-411ed. We further investigated the effect of miR-411ed in vivo, observing a reduction in tumor size with miR-411ed in combination with Osimertinib but not with miR-411ed or Osimertinib treatment alone, confirming the effectiveness of miR-411ed in TKI response.

(BioRxiv All) A multi-omics approach to identify the impact of miR-411ed on NSCLC TKI resistance: Tyrosine Kinase inhibitors (TKIs) are widely used as effective chemotherapeutic agents for treating patients with EGFR-mutated NSCLC. Unfortunately, after treatment, patients… #BioRxiv #MassSpecRSS

A stage-resolved map of dynamic septin interactions required for infection by the rice blast fungus Septin GTPases are essential cytoskeletal regulators that organize membranes and scaffold protein complexes to control cytokinesis, polarity, and morphogenesis. How septins execute these functions remains poorly understood, and comprehensive, stage-resolved interaction maps are lacking. Here, we define a quantitative, time-resolved septin interactome in the rice blast fungus Magnaporthe oryzae using immunoprecipitation coupled to mass spectrometry. We map more than 350 interactors of septins Sep3, Sep4, Sep5 and Sep6, revealing a dynamic network required for appressorium-mediated plant infection. Beyond canonical roles in cytoskeletal organisation and polarity, septins associate with proteins linked to membrane remodelling, metabolism, and virulence, deployed during host invasion. Integration with ultra-high-throughput yeast two-hybrid analysis defines a high-confidence septin interactome and identifies previously uncharacterised factors, including Msi1, a BAR domain protein required for invasive growth. Together, these findings establish septins as dynamic organisers of infection-related processes and provide a framework for understanding how cytoskeletal scaffolds coordinate fungal pathogenesis.

(BioRxiv All) A stage-resolved map of dynamic septin interactions required for infection by the rice blast fungus: Septin GTPases are essential cytoskeletal regulators that organize membranes and scaffold protein complexes to control cytokinesis, polarity, and morphogenesis.… #BioRxiv #MassSpecRSS

FAM134B isoform 2/RETREG1-2 defines a calnexin-TOLLIP-coupled ER-phagy pathway that restricts Ebola virus glycoprotein and is antagonized by VP40 through macro-autophagy Selective autophagy of the endoplasmic reticulum (ER-phagy) is critical for ER proteostasis and host defense, yet how ER quality-control pathways interface with ER-phagy to restrict viral glycoproteins remains poorly defined. Previously, the 1st known ER-phagy receptor gene RETREG1 (RETR1)/FAM134B gene was reported to restrict Ebola virus (EBOV) replication in vivo by inhibiting the viral glycoprotein (GP) and viral protein 40 kDa (VP40) expression, but this mechanism remains unknown. Here, we identify the truncated RETR1/FAM134B isoform 2 (RETR1-2), but not its full-length protein RETR1, as an ER-phagy receptor that targets EBOV-GP for degradation. RETR1-2 broadly triggers GP degradation across ebolavirus species but not Marburg virus and inhibits EBOV replication. Mechanistically, RETR1-2 recognizes EBOV-GP via its luminal domain, undergoes GP-induced oligomerization, and directs GP-containing ER membranes to lysosomes through canonical macro-autophagy. Using unbiased mass spectrometry, we identified TOLLIP as the key cytoplasm adaptor for RETR1-2, which also requires cooperation with the ER chaperone calnexin for EBOV-GP degradation. Notably, the PI3P-binding C2 domain of TOLLIP mediates its interaction with RETR1-2, and the EBOV-GP degradation occurs independently of ubiquitination, revealing an unexpected role for TOLLIP in ER-phagy. Furthermore, EBOV-VP40 antagonizes this pathway by selectively targeting RETR1-2 for macroautophagic degradation independently of TOLLIP, thereby restoring GP expression and viral infectivity. Nevertheless, RETR1-2 reciprocally degrades VP40 via a similar mechanism. Together, these findings define a calnexin-TOLLIP-RETR1-2 axis that links ER quality control to ER-phagy-mediated antiviral restriction and uncover a reciprocal host-virus arms race centered on selective macro-autophagy.

(BioRxiv All) FAM134B isoform 2/RETREG1-2 defines a calnexin-TOLLIP-coupled ER-phagy pathway that restricts Ebola virus glycoprotein and is antagonized by VP40 through macro-autophagy: Selective autophagy of the endoplasmic reticulum (ER-phagy) is critical for ER proteostasis… #BioRxiv #MassSpecRSS

The metabolome and proteome of stem cell-derived human primordial germ cells: a multi-omics approach Primordial germ cells (PGCs) are the population of cells that, in the human embryo, specify day 12 post-fertilization, and form the precursor cells for the future egg or sperm cells. Although in vitro differentiation of PGCs from human stem cells has been achieved, these primordial germ cell-like cells (hPGCLCs) fail to further mature. The reason for this is unclear. Previous studies in mice revealed that several specific metabolic changes occur during the maturation of these cells, which are essential for their developmental progress. However, very little is known about the metabolic profile of human primordial germ cells. In the severe scarcity of human PGCs, hPGCLCs serve as a research model to study PGC formation. To investigate this, we differentiated hPGCLCs using induced-pluripotent stem cells and performed a mass spectrometry analysis to establish their metabolome and proteome. These cells revealed distinct metabolic profile, with changes particularly at the proteome level. This included a shift between canonical and non-canonical citric acid cycle in hPGCLC, downregulation of late-stage glycolysis and reduction of nucleotide de novo synthesis. By providing an integrative map of these metabolic networks, we aim to provide insight on the influence of metabolism on human PGC development that could help improve methods for in vitro differentiation and maturation hPGCLCs.

(BioRxiv All) The metabolome and proteome of stem cell-derived human primordial germ cells: a multi-omics approach: Primordial germ cells (PGCs) are the population of cells that, in the human embryo, specify day 12 post-fertilization, and form the precursor cells for the… #BioRxiv #MassSpecRSS

Ovarian extracellular matrix mechanics regulate oocyte-follicle interactions during female reproductive aging Female reproductive aging is associated with ovarian functional decline, leading to infertility. During aging, biochemical and biophysical changes in the ovarian extracellular matrix (ECM) occur, yet how these properties affect follicle growth and oocyte quality remains poorly understood. Here we describe spatiotemporal changes in the ovarian ECM with age using mass spectrometry, immunohistochemistry, and nanoindentation. While follicle stiffness remains unchanged, stromal matrix remodeling is associated with a ~2.5-fold increase in stiffness. To understand how this increase in stromal stiffness affects age-related follicular dysfunction, isolated young follicles were cultured in soft and stiff hydrogels mimicking young and aged ovarian stromal stiffness, respectively. Higher stiffness leads to a decrease in granulosa cell (GC) proliferation, oocyte quality, and GC-oocyte interactions mediated via transzonal projections (TZPs). RNA-seq revealed TGF-{beta} signaling as a major pathway affected by stiffness, and activation of TGF-{beta} signaling through Mongersen treatment rescued TZP formation and oocyte quality in stiff matrix. These findings provide mechanistic insight into how changes in ECM mechanics contribute to ovarian aging functional decline and reveal potential therapeutic targets to counter fertility loss associated with tissue aging and fibrosis.

(BioRxiv All) Ovarian extracellular matrix mechanics regulate oocyte-follicle interactions during female reproductive aging: Female reproductive aging is associated with ovarian functional decline, leading to infertility. During aging, biochemical and biophysical changes in… #BioRxiv #MassSpecRSS