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

Sex-specific remodeling of the tRNA epitranscriptome in Alzheimers disease tRNA modifications are critical regulators of RNA stability, decoding fidelity, and cellular stress adaptation, yet their contribution to human neurodegenerative disease remains poorly understood. Beyond their established functions in translational control, emerging evidence shows that RNA modifications influence neurogenesis, neurodevelopment, neuronal function, brain-cell differentiation, and cellular plasticity. Consequently, dysregulation of these molecular processes is increasingly recognized as a mechanistic contributor to neurodegenerative disorders. Alzheimers disease (AD), characterized by amyloid pathology, synaptic dysfunction, and progressive neuronal loss, has recently been linked to disturbances in RNA metabolism, suggesting that alterations in the epitranscriptome may represent an underexplored dimension of AD pathophysiology. Here, we systematically profiled the tRNA epitranscriptome across cellular and animal models of AD, as well as in human postmortem brain tissue from non-demented controls and AD patients, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method enables highly sensitive quantification of RNA modifications, with limits of detection in the low femtomole range. Across our models, we identified a conserved yet sex-specific remodeling of the tRNA modification landscape in AD. Because therapeutic options and early diagnostic tools for AD remain limited, we leveraged these findings to develop a tRNA-centered RNA-modification score that integrates both nucleobase-specific modification patterns and neuropathological disease severity into a quantitative metric. Together, our findings identify the tRNA epitranscriptome as a unifying molecular sex-specific signature of AD, linking disease pathology and sex to impaired RNA metabolism. This line of research opens a new path toward establishing early biomarkers or diagnostic tools for AD.

(BioRxiv All) Sex-specific remodeling of the tRNA epitranscriptome in Alzheimers disease: tRNA modifications are critical regulators of RNA stability, decoding fidelity, and cellular stress adaptation, yet their contribution to human neurodegenerative disease remains poorly… #BioRxiv #MassSpecRSS

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Single Cell-Type Spatial Proteomics Uncovers Regional Heterogeneity of Astrocytes Astrocytes are a subset of glial cells in the central nervous system (CNS) that support numerous processes essential for brain function. Their functional diversity is thought to arise from specialized subpopulations with distinct molecular profiles. Although single-cell and single-nucleus RNA sequencing (scRNA-seq and snRNA-seq) have greatly advanced our understanding of astrocyte transcriptomic heterogeneity, mRNA abundance does not always correlate with protein levels because of post-transcriptional and translational regulation. Therefore, studying protein profiles remains essential to accurately capture astrocyte functional states and heterogeneity. Here, we used Microscoop Mint, a microscopy-guided spatial proteomics platform that integrates subcellular, region-specific sample preparation with LC-MS/MS-based mass spectrometry, enabling direct protein profiling of astrocytes in paraformaldehyde-fixed, optimal cutting temperature (OCT)-embedded mouse brain tissue. By applying this approach, we uncovered distinct regional-associated astrocyte proteomic signatures in the cerebral cortex and hippocampus and selected novel candidate protein markers for subsequent validation by immunofluorescence. Notably, MINK1 and PLEKHB1 showed preferential expression in hippocampal and cortical astrocytes, respectively, highlighting their potential as region-specific astrocyte markers. Overall, this strategy enables high-precision, unbiased spatial proteomic discovery at subcellular resolution, providing a powerful framework for linking molecular diversity to functional specialization in astrocyte biology.

(BioRxiv All) Single Cell-Type Spatial Proteomics Uncovers Regional Heterogeneity of Astrocytes: Astrocytes are a subset of glial cells in the central nervous system (CNS) that support numerous processes essential for brain function. Their functional diversity is thought to… #BioRxiv #MassSpecRSS

Inositol phosphates, pyrophosphates and the genes involved in their turnover in the streptophyte green alga Chara braunii Inositol phosphates (InsPs) and inositol pyrophosphates (PP-InsPs) are conserved signalling molecules, but their evolutionary origin and diversification in the green lineage remain poorly understood. Here we investigated the InsP network in the streptophyte alga Chara braunii, a key lineage close to the origin of land plants. Using capillary electrophoresis-electrospray ionization mass spectrometry, we detected a broad spectrum of InsP and PP-InsP species from InsP3 to InsP8, including multiple positional isomers. Developmental profiling across dormant oospores, young thalli and mature thalli revealed extensive metabolic remodeling, with InsP6 as the dominant metabolite and distinct stage-dependent changes in lower InsPs and pyrophosphorylated species. Multiple PP-InsP5 and (PP)2-InsP4 isomers were identified, together with an unassigned additional InsP8-like signal, indicating further pathway complexity. Bioinformatic analyses identified candidate homologs of major InsP metabolic enzymes, supporting the presence of an enzymatic framework for InsP synthesis and turnover similar to land plants. Environmental perturbation revealed isomer-selective effects: prolonged light and dark phases strongly affected the accumulation of specific InsP5 and PP-InsP5 isomers, with 1-PP-InsP5 emerging as the most stimulus-responsive pyrophosphate species, whereas heat stress preferentially reduced 4-PP-InsP5. Together, these findings show that a structurally complex and environmentally responsive InsP network was already established in streptophyte algae before the emergence of land plants.

(BioRxiv All) Inositol phosphates, pyrophosphates and the genes involved in their turnover in the streptophyte green alga Chara braunii: Inositol phosphates (InsPs) and inositol pyrophosphates (PP-InsPs) are conserved signalling molecules, but their evolutionary origin and… #BioRxiv #MassSpecRSS

ER-Lysosome Cholesterol Exchange Regulates Lysosomal Motility Through mTOR-Dependent LAMTOR1 Phosphorylation The subcellular distribution of lysosomes, the main degradative organelles of mammalian cells, responds to metabolic cues in a highly dynamic way. While lysosomal positioning due to amino acid levels is well-characterized, cholesterol-dependent regulation of lysosomal motility is incompletely understood. We explored impaired lysosomal cholesterol export using a mass spectrometry-based multi-OMICs approach, identifying widespread reallocation of resources and signaling pathway modulation. We identified increased phosphorylation at LAMTOR1 serine 56 in response to cholesterol level perturbations. We demonstrate that this phosphorylation site is sufficient to disrupt Rag GTPases/SLC38A9 binding to the Ragulator complex, inhibiting canonical mTORC1 and facilitating binding of BORC, therefore promoting lysosomal retrograde movement. LAMTOR1 S56 phosphorylation responds exclusively to depletion of lysosomal limiting membrane cholesterol, is facilitated by mTOR, and presents a negative feedback loop for amino acid independent displacement of Ragulator bound Rag GTPases, limiting canonical mTORC1 activity. Mass spectrometry data are available via ProteomeXchange with identifier PXD073489.

(BioRxiv All) ER-Lysosome Cholesterol Exchange Regulates Lysosomal Motility Through mTOR-Dependent LAMTOR1 Phosphorylation: The subcellular distribution of lysosomes, the main degradative organelles of mammalian cells, responds to metabolic cues in a highly dynamic way. While… #BioRxiv #MassSpecRSS

A lateral linker histone binding mode scaffolds dinucleosome stacking in chromatin fibers Linker histones are essential for chromatin compaction, yet how they contribute to higher-order fiber assembly remains poorly understood. Here, we determined cryo-electron microscopy structures of Arabidopsis dodeca-nucleosome fibers containing distinct H2A/H3 variants and linker histone H1.3, revealing a noncanonical binding mode that a laterally positioned H1.3 connects the acidic patch of one nucleosome and the DNA of the neighboring nucleosome, thereby scaffolding dinucleosomes into two-start chromatin fibers. This noncanonical binding mode is structurally conserved when H1.3 is replaced by Gallus gallus H5. Furthermore, incorporation of H2A.W and H3.3 further induces back-to-back fiber dimerization. Cryo-electron tomography and in vivo cross-linking mass spectrometry analyses support the physiological relevance of H1 lateral engagement. Our findings establish that linker histones act as active architectural scaffolds in higher-order chromatin organization.

(BioRxiv All) A lateral linker histone binding mode scaffolds dinucleosome stacking in chromatin fibers: Linker histones are essential for chromatin compaction, yet how they contribute to higher-order fiber assembly remains poorly understood. Here, we determined cryo-electron… #BioRxiv #MassSpecRSS

Distribution, assembly and mechanism of GluN1/GluN3A excitatory glycine receptors NMDA receptors play key roles in brain development, plasticity and diseases. While glutamate and glycine co-gated GluN2-containing NMDARs have been extensively characterized, little is known regarding GluN3A-containing NMDARs that form receptors gated by glycine only. Here, combining native purification, mass spectrometry, cryo-EM and electrophysiology, we provide key insights on the molecular logic of GluN3A-NMDARs. We demonstrate that native GluN3A receptors account for a sizeable fraction of total NMDARs, are enriched at extrasynaptic compartments in the adult brain, and assemble specifically as diheteromeric GluN1/GluN3A excitatory glycine receptors (eGlyRs) rather than as triheteromeric GluN1/GluN2/GluN3A receptors. The architecture of eGlyRs reveal splayed and loosely packed extracellular domains, strikingly different from conventional GluN1/GluN2 receptors. Through back-and-forth structural and functional validations, we demonstrate how the combined effects of a weak ligand-binding domain (LBD) dimer interface and high intrinsic mobility of the N-terminal domains (NTDs) shape the atypical gating of eGlyRs. These findings illuminate GluN3A-NMDAR physiology and mechanism, with implications for neuronal signaling and pharmacology.

(BioRxiv All) Distribution, assembly and mechanism of GluN1/GluN3A excitatory glycine receptors: NMDA receptors play key roles in brain development, plasticity and diseases. While glutamate and glycine co-gated GluN2-containing NMDARs have been extensively characterized,… #BioRxiv #MassSpecRSS

Beyond Delta Masses: MS Andrea Directly Resolves Combinatorial Peptide Modifications in Open Searches Open modification search (OMS) strategies have gained popularity in mass spectrometry-based proteomics for identification of peptides carrying unknown or unexpected post-translational modifications. However, most OMS search engines report only the overall mass difference between the precursor and the matched peptide and do not explicitly identify or score combinations of multiple modifications at the peptide-spectrum match (PSM) level, leaving the interpretation of mass shifts up to the end user and to using downstream analysis tools. Here, we introduce MS Andrea, a novel OMS search engine developed to directly identify and score combinations of up to four variable modifications per peptide without having to predefine them. MS Andrea uses a sequence tag-based strategy to efficiently filter candidate peptides prior to scoring. Remaining candidates are evaluated using the MS Amanda scoring function, first considering fixed modifications only, followed by a second scoring stage in which combinations of modifications from the Unimod database are considered based on the observed mass difference and matched to the spectrum. We evaluated MS Andrea using phosphopeptide datasets from HeLa cells and Arabidopsis thaliana and compared its performance with the widely used OMS engines MSFragger and Sage. Across datasets, MS Andrea identified the highest number of PSMs at 1 % false discovery rate while achieving comparable peptide-level identifications. Importantly, MS Andrea directly reports modification identities and sites at the PSM level and enables the identification of peptides having up to four variable modifications. Together, these results demonstrate that MS Andrea facilitates more detailed and interpretable characterization of peptide modifications while maintaining competitive identification performance in OMS-based proteomic analyses.

(BioRxiv All) Beyond Delta Masses: MS Andrea Directly Resolves Combinatorial Peptide Modifications in Open Searches: Open modification search (OMS) strategies have gained popularity in mass spectrometry-based proteomics for identification of peptides carrying unknown or… #BioRxiv #MassSpecRSS

Serum proteomics reveals distinct phenotypic signatures to IL-6 blockade between two immunotherapies A recent clinical study tested the effects of two different monoclonal antibodies (mAbs) (siltuximab, anti-IL6; tocilizumab, anti-IL6R) on the fate and function of T-cells in people with type 1 diabetes. While both mAbs affect the response of T-cells to stimulation, they have very different, sometimes opposing mechanisms. Here, we use mass-spectrometry based proteomics to analyze longitudinal serum samples (baseline and two weeks post-treatment) from 20 clinical trial participants to examine the effects of siltuximab and tocilizumab on extracellular vesicles. To accomplish this, serum samples were enriched for extracellular vesicles with Mag-Net and analyzed by LC-MS/MS to identify significantly differentially abundant protein groups and pathways. Proteome analysis confirmed highly reproducible measurements across multiple draw dates. In total, we quantified 3300 protein groups of which 46 protein groups had significantly altered abundance after mAb treatment. Tocilizumab altered pathways associated with proteostasis (neddylation) and pre-notch transcription and translation. Siltuximab altered FCGR activation pathway members. In addition, quantitation of the monoclonal antibody therapies themselves enabled the measurement of the correlation between drug amounts and impacted proteins. Taken together, this work demonstrates the utility of the Mag-Net method to evaluate the impacts of therapeutic interventions on serum extracellular vesicles.

(BioRxiv All) Serum proteomics reveals distinct phenotypic signatures to IL-6 blockade between two immunotherapies: A recent clinical study tested the effects of two different monoclonal antibodies (mAbs) (siltuximab, anti-IL6; tocilizumab, anti-IL6R) on the fate and function… #BioRxiv #MassSpecRSS

In-source fragmentation in mass spectrometry-based proteomics: prevalence, impact, and strategies for mitigation Peptide-level analyses are becoming increasingly popular in mass spectrometry-based proteomics and are being applied, for example, in immunopeptidomics, structural proteomics, and analyses of post-translational modifications. In such analyses, peptides that are not biologically meaningful but instead arise as artifacts prior to mass spectrometry analysis pose the risk of data misinterpretation. Here, we describe an approach based on retention time analysis and precise chromatographic peak matching to identify peptides generated by in-source fragmentation (ISF), which occurs between chromatographic separation of peptide mixtures and the first mass filter of a tandem mass spectrometer (MS). To understand the prevalence and properties of ISF, we generated 13 proteomics datasets and analyzed them along with additional 25 previously published datasets spanning a broad range of sample types, MS, and proteomics approaches including classical bottom-up proteomics, immunopeptidomics, structural proteomics, and phosphoproteomics. We found that, in typical trypsin-digested samples on average 1 % of fully-tryptic peptides and 22 % of semi-tryptic peptides originated from ISF. However, we observed large variations between datasets, and in-source fragments exceeded, in some cases, a third of the total peptide identifications. The extent of ISF was dependent on the peptide sequence, the instrument, method parameters, and sample complexity. Although ISF did not impair relative quantification across samples, it generated peptides that could be misinterpreted qualitatively, inflated peptide identifications, and comprised up to 37 percent of peptides shorter than 9 amino acids in immunopeptidomics datasets. We propose that, for peptide-centric applications, our open-source ISF detection approach be used to re-annotate peptides generated by ISF and remove them to avoid misinterpretation of data. ISF is an increasing concern with improving mass spectrometers, as they enable detection of an ever-increasing number of m/z features, including low abundance features like ISF products. Our work thus addresses a growing issue in proteomics and presents solutions to mitigate the impact of in-source fragment peptides. In the future, improved feature detection algorithms may enable elucidation of new ISF patterns affecting side chains that have been missed so far, which could contribute to explaining the vast space of as-yet unannotated proteomics data.

(BioRxiv All) In-source fragmentation in mass spectrometry-based proteomics: prevalence, impact, and strategies for mitigation: Peptide-level analyses are becoming increasingly popular in mass spectrometry-based proteomics and are being applied, for example, in… #BioRxiv #MassSpecRSS

The phosphoS655 Alzheimer's Amyloid Precursor Protein (APP) interactome in neuronal differentiation The Alzheimer's Amyloid Precursor Protein (APP) has determinant roles in neuronal development and function, both in its full-length conformation and as some of its proteolytic peptides, particularly secreted (s)APPa. Given that APP phosphorylation tightly regulates its trafficking, proteolysis, and protein-protein binding, it consequently affects several APP functions. The S655 residue, located in the basolateral sorting motif YTSI at APP C-terminus has been observed to be phosphorylated in mature full-length APP and its C-terminal fragments. Previously observed to modify APP's protein interactions, resulting in altered endolysosomal trafficking, andincreased half-life and sAPPa generation, phosphoS655 APP has potential to modulate APP-mediated neuronal differentiation. To study the phosphoS655 differential interactome relevant for neuronal differentiation, SH-SY5Y cells expressing Wt or S655 phosphomutants APP-GFP were differentiated at two time points. APP-GFP and their respective interacting partners were immunoprecipitated using GFP-trap, and interactors identified by mass spectrometry. Both dephospho and phosphoS655 interactomes were generally enriched in similar processes, primarily RNA processing and translation, as well as signal transduction, metabolism, and cytoskeleton remodeling. The smaller phosphoS655 interactome contributes for functional specialization via binding to e.g. FUBP3, ELAVL4, ATXN2, Tubulin, INA. Several of these specific binding partners are known to promote neurite outgrowth and likely underlie our experimental observation that phosphoS655 APP promotes neuritogenesis, particularly the formation of longer neuritic extensions. These results are not only important for the body of knowledge on this Alzheimer's disease core protein, but may also aid in future therapies against this disease.

(BioRxiv All) The phosphoS655 Alzheimer's Amyloid Precursor Protein (APP) interactome in neuronal differentiation: The Alzheimer's Amyloid Precursor Protein (APP) has determinant roles in neuronal development and function, both in its full-length conformation and as some of… #BioRxiv #MassSpecRSS

SPEx: Compartment-Resolved Proteomics via Expansion Microscopy-Guided Microdissection Cells contain different organelles and compartments that are essential for cellular function and life. These organelles and compartments need to communicate to assess cellular state in a changing environment, adapt to the new situation, and also to ensure functionality and homeostasis. Moreover, organization and communication differ between cell types. However, our knowledge about these changes is still rather scarce. Subcellular spatial proteomics aims to fill this knowledge gap. While proximity labeling techniques represent a great advance, they do not provide precise spatial resolution. To overcome this limitation, we developed SPEx (Subcellular spatial Proteomics coupled to Expansion), in which we first expand cells about 10-fold, laser micro-dissect regions of interests and then perform mass spectrometry-based proteomics on these samples. We demonstrate the effectiveness of SPEx by determining the proteome of the Golgi, the nucleus and nucleoli. Satisfyingly, we also identify novel components of these organelles. Combining inexpensive already existing technologies makes SPEx readily usable by the wider scientific community.

(BioRxiv All) SPEx: Compartment-Resolved Proteomics via Expansion Microscopy-Guided Microdissection: Cells contain different organelles and compartments that are essential for cellular function and life. These organelles and compartments need to communicate to assess cellular… #BioRxiv #MassSpecRSS

Hyperosmolar stress promotes the release of small extracellular vesicles containing metabolic proteins from corneal epithelial cells Purpose: Hyperosmolar stress (HOS) is a major contributor to corneal epithelial cell damage in dry eye disease. We have previously shown that HOS damages mitochondria and impairs cell metabolism in corneal epithelial cells. Small extracellular vesicles (sEVs) are cell-derived lipid envelopes that are present in all body fluids, including tears. Prior studies suggest that sEV release and composition may be linked with changes in cell metabolism. In this study, we tested the effects of HOS on sEV release and composition, and found that sEV cargo may reflect early, underlying changes in dry eye disease. Methods: Telomerase-immortalized human corneal epithelial (hTCEpi) cells were treated with 450 mOsm NaCl for five days to induce chronic HOS. sEVs were isolated using differential centrifugation followed by iodixanol density gradient flotation. Particle number was determined using Nanoparticle Tracking Analysis (NTA). Mass spectrometry was used to assess the sEV proteome, and selected proteins were validated by immunoblot. Proteome pathways were analyzed using KEGG and CORUM. Results: Pathway analysis revealed an increase in metabolic proteins and proteasome components in sEV cargo released from hTCEpi cells exposed to HOS. These proteins were increased more than fourfold in HOS-sEVs. Examination of proteins involved in the endosomal pathway and NTA further confirmed an increase in HOS-sEV release. Conclusion: Our findings suggest a potential mechanism whereby corneal epithelial cells exposed to HOS retain proteins involved in maintaining tissue integrity, while simultaneously releasing unneeded proteins involved in cell metabolism. The presence of metabolic proteins in sEVs may serve as early indicators of dry eye disease.

(BioRxiv All) Hyperosmolar stress promotes the release of small extracellular vesicles containing metabolic proteins from corneal epithelial cells: Purpose: Hyperosmolar stress (HOS) is a major contributor to corneal epithelial cell damage in dry eye disease. We have… #BioRxiv #MassSpecRSS

Optimization of Retinoid Detection in Cerebrospinal Fluid Using Liquid Chromatography Mass Spectrometry Introduction: Retinoids are bioactive vitamin A derivatives that regulate cellular differentiation and gene expression, yet their reliable quantification remains challenging due to low abundance, structural isomerism, and sensitivity to ionization conditions while handling. Objectives: In this study, we performed a systematic optimization of liquid chromatography -- mass spectrometry (LC-MS)-based detection of retinoids across tissues and biofluids. Methods: Chromatographic separation, adduct formation, ionization parameters, fragmentation behavior, and extraction procedures were evaluated in an integrated workflow. Results: Chromatographic conditions influenced not only retention time but also the ionic species detected, affecting precursor selection for MS2 analysis. Retinoids exhibited compound-dependent responses to electrospray ionization and collision energy, requiring tailored acquisition parameters. Extraction experiments demonstrated differential recovery among retinoid classes and revealed matrix-dependent behavior, indicating that protocols used for tissues cannot be directly transferred to low-abundance biofluids. Using optimized conditions, retinoids were detected in mouse cerebrospinal fluid (CSF) at concentrations approaching the analytical detection limit, where MS2 confirmation was necessary for reliable identification. Conclusion: Together, our results provide a framework for reproducible retinoid profiling across biological matrices and enables comparative studies of retinoid biology in low-volume and low-abundance biofluids.

(BioRxiv All) Optimization of Retinoid Detection in Cerebrospinal Fluid Using Liquid Chromatography Mass Spectrometry: Introduction: Retinoids are bioactive vitamin A derivatives that regulate cellular differentiation and gene expression, yet their reliable quantification… #BioRxiv #MassSpecRSS

GAPMs form a heterotrimeric complex bridging the gliding machinery and the cytoskeleton across Plasmodium species Apicomplexan parasites, such as malaria-causing Plasmodium spp., use a specialised actomyosin motor system known as the glideosome to drive movement through host tissue and invade host cells. This system is anchored to the inner membrane complex (IMC), a series of flattened vesicles located beneath the plasma membrane, and thought to be linked to the underlying cytoskeleton by the GAPM protein family. However, it is not known how these GAPM proteins are localised across the Plasmodium life cycle, and whether different family members function alone or together. Here, we show that in two Plasmodium species GAPM2 is an IMC component whose recruitment and organisation are tightly coordinated with nuclear and cytoskeletal dynamics during parasite replication and differentiation. We find that the GAPM2 interactome remodels between asexual and sexual stages using mass spectrometry. To understand the molecular relationship between three GAPM paralogues, we solved a cryo-electron microscopy structure of the GAPM complex. This revealed an obligate heterotrimeric architecture that forms an asymmetric platform, likely to serve as a docking interface for other components of the glideosome. Finally integrating our GAPM heterotrimer structure with mass spectrometry data allowed us to propose a unified structural model of the glideosome that is conserved across apicomplexan parasites.

(BioRxiv All) GAPMs form a heterotrimeric complex bridging the gliding machinery and the cytoskeleton across Plasmodium species: Apicomplexan parasites, such as malaria-causing Plasmodium spp., use a specialised actomyosin motor system known as the glideosome to drive movement… #BioRxiv #MassSpecRSS

Host cell remodeling via cyclin dependent kinases drives Ebola virus replication and transcription Ebola virus (EBOV) causes re-emerging outbreaks of viral hemorrhagic fever with high case-fatality rates. As a negative-sense single stranded RNA virus, EBOV depends on its RNA-dependent RNA polymerase (L protein) to transcribe and replicate the viral genome. This takes place in cytoplasmic inclusion bodies that form following host cell remodeling involving yet unknown signaling pathways. Here, using mass spectrometry-based (phospho-)proteomics, we profiled global protein abundance and site-specific phosphorylation in HEK293T cells expressing an EBOV minigenome system. Our data reveal EBOV-induced rewiring of the host proteome and phosphorylation signaling landscape, including perturbations in cell cycle control, cytoskeletal organization, innate immune regulation, and DNA damage response. Kinase network analysis revealed that Hippo pathway kinases and especially cyclin dependent kinases like CDK2 are central drivers of EBOV replication and transcription. The functional necessity of these signaling pathways is demonstrated via inhibition of CDK family kinases with small molecule inhibitors, which halted EBOV minigenome replication and transcription when administered in the low micromolar range, demonstrating that these pathways represent putative antiviral targets.

(BioRxiv All) Host cell remodeling via cyclin dependent kinases drives Ebola virus replication and transcription: Ebola virus (EBOV) causes re-emerging outbreaks of viral hemorrhagic fever with high case-fatality rates. As a negative-sense single stranded RNA virus, EBOV… #BioRxiv #MassSpecRSS

Molecular Dosimetry of DNA Adducts in Mice Exposed to Ethylene Oxide Ethylene oxide (EtO) is a highly reactive industrial chemical and classified as a known human carcinogen with a putative mutagenic mode of action (MOA). Its genotoxic potential is primarily mediated through alkylation of DNA, resulting in the formation of the mutagenic adduct O6-(2-hydroxyethyl)-2'-deoxyguanosine (O6-HE-dG). The N7-(2-hydroxyethyl)guanine (N7-HE-G) adduct is formed in greater abundance and is generally considered to be non-mutagenic. However, dose-response relationships of these DNA adducts, particularly at low inhalation exposure levels (i. e., below 3 ppm), remain unknown. These data are necessary to inform the biological plausibility of different statistical dose-response models that have been applied to human or animal data used for cancer risk assessment. In the present study, male and female B6C3F1 mice were exposed to EtO (0, 0.05, 0.1, 0.5, 1, 50, 100, and 200 ppm) 6 hours/day for 28 consecutive days. Immediately following the last exposure, DNA was extracted from lung, liver, bone marrow, and mammary gland, and further utilized to measure DNA adduct levels using highly sensitive mass spectrometry platforms. N7-HE-G was detected in all tissues and exposure groups, showing linear dose-response relationships in the low-dose range ([≤]1 ppm) and increased sharply and exposure-disproportionately in the high-dose range ([≥]50 ppm). Despite a very low limit of detection, O6-HE-dG, in contrast, was not detected at exposures

(BioRxiv All) Molecular Dosimetry of DNA Adducts in Mice Exposed to Ethylene Oxide: Ethylene oxide (EtO) is a highly reactive industrial chemical and classified as a known human carcinogen with a putative mutagenic mode of action (MOA). Its genotoxic potential is primarily… #BioRxiv #MassSpecRSS

Identification of a somatic H3K23me3 methyltransferase SET-19 in C. elegans Histone methylation plays essential roles in modulating chromatin organization and gene expression. H3K23 methylation is a conserved histone modification, yet its biological roles and the enzymes responsible for its deposition remain poorly understood. Here, we show that the loss of set-19 leads to a pronounced reduction in H3K23 methylation in C. elegans, as revealed by quantitative mass spectrometry, western blotting, and immunofluorescence staining. In vitro biochemical assays show that recombinant SET-19 proteins purified from E. coli directly catalyze H3K23 methylation. Genome-wide chromatin immunoprecipitation assays reveal that H3K23me3 is enriched at heterochromatic regions and that loss of set-19 alters H3K23me3 levels, accompanied by derepression of gene expression. Genetic analyses indicate that SET-19 is dispensable for both germline and somatic RNAi as well as transgenerational epigenetic inheritance of RNAi. SET-19 is predominantly expressed in somatic cells and specifically mediates H3K23me3 deposition in somatic tissues. The loss of set-19 causes a developmental delay without affecting fertility. Together, our results identify SET-19 as a somatic H3K23 methyltransferase and link H3K23me3 to gene repression in C. elegans.

(BioRxiv All) Identification of a somatic H3K23me3 methyltransferase SET-19 in C. elegans: Histone methylation plays essential roles in modulating chromatin organization and gene expression. H3K23 methylation is a conserved histone modification, yet its biological roles and… #BioRxiv #MassSpecRSS

Uncovering zebrafish embryonic proteome dynamics across 16 time points during the first 24 hours of development Defining how proteins change over developmental time is amenable to studies deciphering regulatory genetic networks in vertebrate development, biology, and pharmacology. In an approach toward such quantitative studies of dynamic network behavior, we produced an atlas using the mass spectrometry-based method to investigate protein expression changes across 16 time points from the zygote to the early pharyngula stage zebrafish embryos. We systematically summarize 8 clusters for interrogating changes in protein expression associated with the development of zebrafish embryos. Specifically, we identified a class of zinc finger-related transcription factors primarily located on the long arm of chromosome 4, which are highly expressed during zygotic genome activation. Furthermore, we highlight the power of this analysis to assign developmental stage-specific expression information to chromosomes and tissues. Time-resolved analyses reveal significant discordance between differential transcript and protein expression, whereas no time lag is observed for proteins involved in stable and fundamental biological processes, such as metabolism (e.g., Ppt2a and Gatm), cytoskeletal organization (e.g., Col18a1), and the translation machinery (e.g., Eif4enif1). This atlas offers high-resolution and in-depth molecular insights into zebrafish development, providing a resource for developmental biologists to generate hypotheses for functional analysis of proteins during early vertebrate embryogenesis.

(BioRxiv All) Uncovering zebrafish embryonic proteome dynamics across 16 time points during the first 24 hours of development: Defining how proteins change over developmental time is amenable to studies deciphering regulatory genetic networks in vertebrate development,… #BioRxiv #MassSpecRSS

Single-cell proteomics reveals proteome remodeling and cellular heterogeneity during NGF-induced PC12 neuronal differentiation Single-cell proteomics (SCP) enables direct measurement of cellular heterogeneity during dynamic biological processes. Here, we applied an SCP workflow to investigate proteome diversity during nerve growth factor (NGF)-induced differentiation of PC12 cells. Differentiated PC12 cells are highly adherent and prone to aggregation, complicating single-cell sample preparation. To address this challenge, sample handling was optimized using gentle dissociation, anti-adhesive conditions, and rapid processing immediately prior to cell isolation. Individual cells were deposited using a refined thermal inkjet (TIJ) dispensing system, enabling accurate single-cell placement with minimal sample loss. Inclusion of the mild nonionic surfactant DDM improved recovery of membrane-associated and other low-solubility proteins. Coupled with high-sensitivity liquid chromatography-ion mobility-mass spectrometry, this workflow consistently quantified approximately 2,000-3,000 proteins per cell across differentiation stages. Single-cell proteomic profiles acquired over the differentiation time course revealed clear separation between undifferentiated and NGF-treated cells by Day 6. At later stages (Days 4-6), cells further partitioned into two distinct subpopulations with protein expression patterns not evident in bulk measurements. Dimensionality reduction and non-negative matrix factorization identified multiple proteomic states coexisting within the same differentiation stages, characterized by coordinated differences in pathways related to intracellular trafficking, protein translation, and neuronal structural organization. Together, these results show that while global proteome remodeling during PC12 differentiation is captured in both bulk and single-cell data, single-cell proteomics uniquely resolves functionally distinct cellular subpopulations that are masked in population-averaged analyses.

(BioRxiv All) Single-cell proteomics reveals proteome remodeling and cellular heterogeneity during NGF-induced PC12 neuronal differentiation: Single-cell proteomics (SCP) enables direct measurement of cellular heterogeneity during dynamic biological processes. Here, we applied… #BioRxiv #MassSpecRSS

Quaternary structure and activity of glutamate dehydrogenase are regulated by reversible S-palmitoylation and mitochondrial acyl-protein thioesterases. Glutamate dehydrogenase (GDH) is a key mitochondrial enzyme that catalyzes the reversible oxidative deamination of glutamate to -ketoglutarate, thereby linking amino acid and carbohydrate metabolism. GDH forms catalytically active hexamers and is regulated by various allosteric modulators, including ADP and GTP. Here, we demonstrate that GDH undergoes auto-palmitoylation in the presence of palmitoyl-CoA, leading to a dose-dependent inhibition of enzymatic activity. Using acyl-PEG exchange assays and mass spectrometry, we show that GDH monomers are predominantly mono-palmitoylated, with modification detected at multiple cysteine residues, including Cys55, Cys115, and Cys197, among the six cysteines in the mature enzyme. Blue Native PAGE analysis revealed that palmitoylation disrupts the native hexameric assembly of mammalian GDH, which is organized as a dimer-of-trimers, promoting dissociation into dimers. Importantly, this modification is reversible, as incubation with mitochondrial acyl-protein thioesterases 1 (APT1) and, to a lesser extent, /{beta} hydrolase domain 10 (ABHD10) restores both the hexameric structure and enzymatic activity. The modified Cys55 residues are positioned near the trimer interface, providing a mechanism by which palmitoylation could prevent hexamer formation, whereas Cys115 and 197 may destabilize individual trimers. These findings establish S-palmitoylation as a novel regulatory mechanism for GDH, linking mitochondrial lipid metabolism to the reversible control of a central metabolic enzyme.

(BioRxiv All) Quaternary structure and activity of glutamate dehydrogenase are regulated by reversible S-palmitoylation and mitochondrial acyl-protein thioesterases.: Glutamate dehydrogenase (GDH) is a key mitochondrial enzyme that catalyzes the reversible oxidative… #BioRxiv #MassSpecRSS

Volatile emissions from diverse estuarine bacteria share core compounds with a subset of strain-specific, low abundance compounds Biogenic volatile organic compounds (BVOCs) are gases that influence atmospheric chemistry, nutrient cycling, and species interactions, yet the contribution of heterotrophic marine bacteria to marine BVOC emissions remains poorly constrained. In addition, the extent to which the volatilome is linked to bacterial phylogeny is unknown. Here, we characterize the volatilome of 16 heterotrophic bacterial strains isolated from Baltic Sea surface water, spanning Alphaproteobacteria, Gammaproteobacteria, Betaproteobacteria, Bacteroidota, and Actinomycetes. Headspace BVOCs were quantified under standardized growth conditions using Proton Transfer Reaction Time-of-Flight Mass Spectrometry (PTR-TOF-MS). A broadly overlapping bacterial volatilome was identified, with compound composition and proportional abundance similar across many strains, irrespective of phylogeny. Namely, most strains shared a core set of abundant compounds with a subset of strain-specific, low abundance compounds. Acetone accounted for more than 50% of the emissions in most volatilomes. The remaining fraction of emissions were primarily comprised of other low-molecular-weight oxygenated compounds. Interestingly, two strains demonstrated strain-specific emission patterns, significantly diverging from the group in their emission rate and compound composition. Together, these findings suggest that marine heterotrophic bacteria may contribute a broadly conserved collection of BVOCs to the ocean-atmosphere interface, highlighting their role as a widespread source of trace gases in marine ecosystems.

(BioRxiv All) Volatile emissions from diverse estuarine bacteria share core compounds with a subset of strain-specific, low abundance compounds: Biogenic volatile organic compounds (BVOCs) are gases that influence atmospheric chemistry, nutrient cycling, and species… #BioRxiv #MassSpecRSS

Deletion of astrocyte intermediate filaments GFAP and Vimentin enhances protein synthesis and prevents early synaptic and cognitive dysfunction in a mouse model of Alzheimer's disease In Alzheimer's disease (AD) astrocytes become reactive, displaying hypertrophic morphology, increased expression of intermediate filament proteins GFAP and Vimentin and impaired homeostatic support to neurons. However, the contribution of reactive astrocytes to AD progression, particularly the role of cytoskeletal hypertrophy, remains unclear. Here, we investigate whether astrocyte intermediate filaments actively contribute to early AD progression. We show that astrogliosis appears as early as at 3 months in APP/PS1 mice, preceding amyloid-{beta} plaque deposition, and is characterized by a strong upregulation of GFAP and Vimentin. Genetic ablation of GFAP and Vimentin attenuated astrogliosis, as evidenced by the absence of hypertrophy of astrocyte processes and restored expression of glutamine synthetase and other proteins altered in reactive astrocytes in AD. Importantly, GFAP and Vimentin deletion prevented cognitive decline in 4-month old male and female mice, independently of amyloid plaque pathology or microglial reactivity. Mass-spectrometry based proteomics of the dorsal hippocampus revealed a downregulation of synaptic proteins and dysregulation of ribosomal and RNA-binding proteins in APP/PS1 mice, both of which were rescued by GFAP and Vimentin deletion. Using astrocyte-specific CRISPR-Cas9-mediated knockout of GFAP and Vimentin, we further demonstrate translation impairments in AD astrocytes, and that GFAP and Vimentin deletion restores this impaired astrocytic translation. Together, our findings identify intermediate filament proteins GFAP and Vimentin as active regulators of astrocyte protein synthesis, and reveal a previously unrecognized mechanism by which reactive astrocytes contribute to early cognitive dysfunction in AD. This highlights these astrocyte intermediate filaments as promising therapeutic targets to counteract reactive astrocyte-driven cognitive decline in the early stages of Alzheimer's disease.

(BioRxiv All) Deletion of astrocyte intermediate filaments GFAP and Vimentin enhances protein synthesis and prevents early synaptic and cognitive dysfunction in a mouse model of Alzheimer's disease: In Alzheimer's disease (AD) astrocytes become reactive, displaying… #BioRxiv #MassSpecRSS

Integrated phytohormone quantification and metabolomics analysis enables new insights into the soil phytohormonome The contribution of soil chemistry to plant growth and resilience, including presence of phytohormones, is increasingly recognized. However, comprehensive characterization of soil phytohormones remains limited by chemical complexity of soil matrices, diversity and low-abundance of metabolites. To enable further discoveries we developed and validated performance of a liquid chromatography-mass spectrometry method with solid phase extraction, integrating targeted and untargeted hormonomic approaches for comprehensive soil phytohormone profiling. Method performance was evaluated for sixteen plant growth-regulating compounds and precursors, including abscisic acid, auxins, cytokinins, gibberellic acid, jasmonic acid, salicylic acid, karrikins, melatonin, serotonin, and tryptophan. The method demonstrated strong linearity (R2 = 0.989-0.999), high sensitivity (limits of detection and quantification 0.1-50.2 and 1.4-167.3 pg on-column, respectively), and acceptable precision (1.3-9.6% intraday; 3.4-34.8% interday). Soil composition had a significant effect on recovery, with recovery being poor in some soils such as clay-rich soils; however, recovery for most phytohormones were within 20% of the matrix-adjusted spiked value. Validation results confirm that the method is suitable for use and was then used to quantify analytes in representative soil types. Integration of untargeted analysis expanded coverage to 250 additional putative phytohormones and hormone-related metabolites, revealing chemical signatures potentially associated with plant community composition. The method is robust across these soils spanning sandy, peat-rich, and clay-rich textures. This approach provides a versatile framework for investigating belowground phytohormone dynamics and their roles in plant physiology, resilience, and soil-plant feedbacks.

(BioRxiv All) Integrated phytohormone quantification and metabolomics analysis enables new insights into the soil phytohormonome: The contribution of soil chemistry to plant growth and resilience, including presence of phytohormones, is increasingly recognized. However,… #BioRxiv #MassSpecRSS

Sequential Dual-Ion MALDI Glycotyping Enables Rapid Phenotypic O-Antigen Typing of Escherichia coli and Shigella Accurate O-antigen typing of Gram-negative bacteria is important for surveillance, outbreak investigation, and quality control of reference strains. However, commonly used typing approaches, including serological agglutination assays and molecular methods, do not always resolve structural variation in expressed O-antigen phenotypes. Here, we describe an improved MALDI glycotyping workflow based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) that enables rapid phenotypic characterization of O-antigen repeating units (RUs). The workflow uses a sequential dual-ion acquisition strategy in which positive-ion spectra are obtained first and negative-ion analysis is triggered only when RU signals are absent, enabling detection of both neutral and acidic O-antigen structures from the same sample spot. Applied to a diverse panel of 71 Escherichia coli and Shigella strains, RU-derived signals were detected in more than 80% of isolates. The approach resolved modification-level structural variation and discriminated isobaric O-antigen phenotypes, enabling scalable phenotypic profiling of O-antigen composition and inference of candidate O-antigen identities from RU-level information. Integration with agglutination testing further revealed discrepancies between archived serotype annotations and expressed O-antigen phenotypes, enabling reassignment of several strains to alternative O-antigen types. Because the workflow can be implemented on MALDI-TOF MS platforms already widely used for microbial identification, this method provides a practical phenotypic complement to conventional O-antigen typing in clinical microbiology laboratories and remains compatible with rapid single-colony MALDI workflows used in routine microbial identification.

(BioRxiv All) Sequential Dual-Ion MALDI Glycotyping Enables Rapid Phenotypic O-Antigen Typing of Escherichia coli and Shigella: Accurate O-antigen typing of Gram-negative bacteria is important for surveillance, outbreak investigation, and quality control of reference strains.… #BioRxiv #MassSpecRSS