Biology subjects

Coon, J. J.

Publications and source records attributed to Coon, J. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Proteome-wide QTL mapping enables gene-protein-phenotype metabolic network construction in a genetically diverse MASLD mouse model

The limited treatment options available for the estimated 38% of adults worldwide affected by Metabolic Dysfunction Associated Steatotic Liver Disease (MASLD) are largely due to an incomplete understanding of the complex molecular networks underlying disease pathogenesis. To dissect the genetic architecture and proteomic regulation underlying MASLD, we generated a genetically diverse mouse cohort through a four-way cross of founder strains with varying susceptibility to liver disease, producing 444 F2 mice with a spectrum of phenotypes and genotypes. Utilizing deep proteomic profiling of the livers of this population, we identified quantitative trait loci (QTL) for 2,652 proteins, spanning over 4,000 unique genomic loci, and distinguished cis- and trans-acting regulatory mechanisms. Integrating proteomic, genomic, and phenotypic data reveals key regulatory loci and candidate proteins influencing disease progression, creating a mineable proteogenomic resource for MASLD research. We utilize this resource to identify the E3 ubiquitin ligase Ubr1 as a candidate central regulator connecting proteostasis and lipid metabolism, with genetic polymorphisms that may alter its abundance and predispose to metabolic dysfunction. This study demonstrates how high-throughput, deep proteome profiling integrated with QTL mapping can reveal complex gene-protein networks governing MASLD susceptibility and progression, offering novel insights for biomarker discovery and therapeutic targeting.

systems biology↗

bioRxiv2026

Rapid Peptide Mapping of Monoclonal Antibodies with Direct Infusion Mass Spectrometry

Peptide mapping is a critical tool for characterizing biotherapeutic proteins and is essential for the development of monoclonal antibody drugs. Here we describe a new direct infusion technology that streamlines peptide mapping data collection and analysis, accelerating the method by up to 100-fold. This method, which we term RaPiD-mAb-MS, combines high-throughput plate-based sample preparation with direct infusion mass spectrometry analysis. RaPiD-mAb-MS allows analysis of 96 samples within [~] 1.5 to 2 hours, routinely achieves >95% sequence coverage, and has been successfully applied to 28 unique antibodies and over 2,000 samples. Here we demonstrate that RaPiD-mAb-MS detects and quantifies oxidation, deamidation, isomerization, glycosylation, and sequence variants with results comparable to conventional LC-MS based methods in a fraction of the time. Further, by eliminating chromatography, data analysis is greatly streamlined and simplified. By allowing for the collection of [~] 1,000 peptide maps per day, RaPiD-mAb-MS is positioned to accelerate all phases of antibody-based drug discovery & development and sets the stage for collection of massive datasets that would allow artificial intelligent prediction of optimal antibody variants and formulations.

biochemistry↗

bioRxiv2026

Co-option of ILF2/3 in primates restrains Alu hyper-editing to enable cell fate transitions

Understanding human specific mechanisms of cell fate control is essential for advancing developmental biology and regenerative medicine. Here, we identify the ILF2/3 complex as a critical regulator of primate cell fate transitions. Using genetically and epigenetically engineered gastruloids and adult stem cells, we show that ILF2/3 is required for gastrulation in primates but not mice, and for differentiation of adult progenitor cells. Mechanistically, ILF2/3 directly binds Alu elements in chromatin-associated RNAs and shields them from ADAR1-mediated adenosine-to-inosine (A-to-I) editing. Acute ILF2/3 degradation increases A-to-I editing at Alu elements, but not murine retrotransposons, leading to aberrant splicing and nonsense-mediated decay of transcripts encoding key chromatin regulators in primate cells. This in turn destabilizes the epigenetic landscape and blocks lineage commitment across all three germ layers. Re-expression of correctly spliced chromatin regulators rescues differentiation defects in ILF2/3-deficient cells, functionally linking Alu editing control to chromatin regulation and cell fate. These findings define an evolutionary mechanism that restrains retrotransposon-associated RNA editing to preserve proteome integrity and enable primate-specific developmental programs.

genetics↗

bioRxiv2026

Atlas of lysine acetylation in the mouse

Lysine acetylation has widespread ramifications from genetic regulatory effects to modulation of enzymatic function. With improved acetyl-lysine enrichment technologies and advances in mass spectrometer speed and sensitivity, we present a comprehensive atlas of the mouse acetylome comprising 17,952 unique lysine acetylation sites across 4,340 proteins and 15 tissues. This resource, which nearly doubles the known mouse acetyl-lysine catalog, shows at least 14% of the acetylome is shared across tissues. We focus our investigation on several acetylated proteins, including ribosomal acetylation and its potential to extend ribosomal half-life in the liver and pancreas. Additionally, we identify a novel acetylation event in the active site of carnitine O-acetyltransferase (Crat) that also mirrors tissue-specific Crat activity. By integrating these data with human pathogenic variants, we identify acetyl-lysine residues on cardiac troponin and homogentisate dioxygenase that likely mimic disease-causing mutations. This resource provides a foundational framework for investigating protein acetylation in metabolic health and disease.

biochemistry↗

bioRxiv2026

Multi-omic analysis reveals nitric oxide dependent remodeling in classically activated macrophages and identifies negative regulation mediated by AKR1A1

Nitric oxide (NO*) is an important signaling molecule in many biological processes, including immune response. During response to classical activation stimuli lipopolysaccharide (LPS) and interferon-{gamma} (IFN{gamma}), macrophages generate NO* via inducible nitric oxide synthase (iNOS). To comprehensively define the effects of NO*, we applied a multi-omic strategy integrating proteomics and transcriptomics to profile murine macrophages across conditions with or without LPS/IFN{gamma}-activation, with or without iNOS expression or exogenous NO* donor treatment. The results revealed NO* has broad, yet selected and controlled, regulatory effects, playing a key role in coordinating the systematic remodeling during macrophage classical activation. Among the proteins that are most suppressed in a NO*-dependent manner, electron transport chain (ETC) is the most enriched. NO* drives complex-specific remodeling of ETC, causing selected downregulation of complex I, II, and IV, through a different combination of transcriptional and post-transcriptional mechanisms for each complex. Functionally, we found NO* is required, but not sufficient, for the strong suppression of cellular respiration upon macrophage activation. Among the most consistently upregulated proteins are many enzymes involved in redox defense. AKR1A1 was identified as a top hit. We found Akr1a1 induction requires both NO* and LPS/IFN{gamma} stimulation. The S-nitroso-CoA reductase activity of AKR1A1 mitigates NO*-driven inhibition of pyruvate dehydrogenase complex by limiting the inhibitory modifications targeting its lipoyl cofactor. Knocking out Akr1a1 causes accelerated remodeling of TCA cycle, dysregulated immunoregulatory metabolite level, and altered functional gene expression and cytokine production at later stage of immune response. Thus, the NO*-dependent upregulation of AKR1A1 forms a negative regulatory loop to fine-tune NO*-mediated metabolic and functional remodeling during immune response. Together, this work provided a systems-level map of NO*-dependent regulation, revealed the crosstalk between NO* and immune signaling, and demonstrated mechanisms providing redox adaptation and precise control of NO*s effects.

immunology↗

bioRxiv2025

Tumor-Derived Polyamines Initiate Fat Wasting in Cancer Cachexia

Cancer-associated cachexia (CC) is a fatal metabolic condition characterized by progressive loss of fat and muscle mass, yet its early molecular drivers remain poorly defined. Here, we identify a polyamine-dependent tumor-adipose crosstalk that triggers adipocyte lipolysis and fat wasting during the pre-cachexia stage, preceding systemic inflammation and muscle atrophy. Cancer-derived polyamines are enriched in extracellular vesicles and promote lipid mobilization via eIF5A hypusination, independent of adrenergic signaling. In preclinical models, polyamine accumulation associates with early fat loss and elevated circulating fatty acids. Clinically, automated CT imaging of newly diagnosed pancreatic cancer patients reveals increased adipose density, reflecting lipolysis, that correlates with circulating polyamine levels and predicts poor survival. These findings support polyamine metabolism as a mechanistic driver and candidate biomarker of early cachexia, providing a framework for early detection and targeted intervention.

cancer biology↗

bioRxiv2025

Orthogonal chemical genomics approaches reveal genomic targets for increasing anaerobic chemical tolerance in Zymomonas mobilis

Genetically-engineered microbes have the potential to increase efficiency in the bioeconomy by overcoming growth-limiting production stress. Screens of gene perturbation libraries against production stressors can identify high-value engineering targets, but follow-up experiments needed to guard against false positives are slow and resource-intensive. In principle, the use of orthogonal gene perturbation approaches could increase recovery of true positives over false positives because the strengths of one technique compensate for the weaknesses of the other, but, in practice, two parallel screens are rarely performed at the genome-scale. Here, we screen genome-scale CRISPRi (CRISPR interference) knockdown and TnSeq (transposon insertion sequencing) libraries of the bioenergy-relevant Alphaproteobacterium, Zymomonas mobilis, against growth inhibitors commonly found in deconstructed plant material. Integrating data from the two gene perturbation techniques, we established an approach for defining engineering targets with high specificity. This allowed us to identify all known genes in the cytochrome bc1 and cytochrome c synthesis pathway as potential targets for engineering resistance to phenolic acids under anaerobic conditions, a subset of which we validated using precise gene deletions. Strikingly, this finding is specific to the cytochrome bc1 and cytochrome c pathway and does not extend to other branches of the electron transport chain. We further show that exposure of Z. mobilis to ferulic acid causes substantial remodeling of the cell envelope proteome, as well as the downregulation of TonB-dependent transporters. Our work provides a generalizable strategy for identifying high-value engineering targets from gene perturbation screens that is broadly applicable. IMPORTANCEEngineering microorganisms to tolerate harsh production conditions stands to increase bioproduct yields of engineered microbes. In this study, we systematically identified Z. mobilis genes that confer resistance or susceptibility to chemical stressors found in deconstructed plant material. We used complementary genetic techniques to cross-validate these genes at scale, providing a widely applicable method for precisely identifying genetic alterations that increase chemical resilience. We discovered genetic modifications that improve anaerobic growth of Z. mobilis in the presence of inhibitory chemicals found in renewable plant-based feedstocks. These results have implications in engineering robust production strains to support efficient and resilient bioproduction. Our methodologies can be broadly applied to understand microbial responses to chemicals across systems, paving the way for developments in biomanufacturing, therapeutics, and agriculture.

genomics↗

bioRxiv2025

Adeno-Associated Virus 2 (AAV2) - induced RPA exhaustion generates cellular DNA damage and restricts viral gene expression

Parvoviruses are single-stranded DNA viruses that have been modified to serve as vehicles for therapeutic transgene delivery in the form of recombinant Adeno-Associated Virus (rAAV2) vectors or rodent parvovirus-derived oncolytic agents. Infection with viruses of the Parvoviridae family induces a cellular DNA Damage Response (DDR) signal that supports virus replication. However, it remains unknown whether rAAV2 vectors or non-replicative AAV2 genomes induce cellular DDR signals, which might be deleterious to the cell. To determine the impact of AAV2/rAAV2 genomes on the integrity of the host chromosome, we have pulsed AAV2/rAAV2 infected cells with BrdU analogs followed by single-molecule imaging of the cellular replisomes and proteomic analysis of the host replication forks. We discovered that non-replicative AAV2/rAAV2 genomes are sufficient to induce replication stress on the host genome, leading to DDR signals in a dose-dependent manner. Moreover, infection with replication-competent AAV2 leads to enrichment of replication stress proteins, DNA repair factors and RNA processing machinery on cellular replication forks. However, neither the AAV2 Inverted Terminal Repeats (ITRs) that are retained in rAAV2s nor empty capsids are sufficient to induce host-cell replication stress. Strikingly, incoming AAV2 genomes associate with the single-stranded DNA binding protein RPA in host cells in a dose-dependent manner, progressively shortening cellular replication forks. These elevated levels of AAV2-induced cellular replication stress eventually leads to accumulation of DDR signals in the nucleus. Chemical inhibition of RPA activity and RNAi-mediated knockdown leads to de-repression of the AAV2 genome, increasing Rep 68/78 gene expression. Ectopic expression of RPA rescues AAV2-induced replication stress. Taken together, our findings suggest that depletion of cellular stores of RPA molecules by competing AAV2 genomes restrict viral gene expression and cause cellular DNA damage. AUTHOR SUMMARYAdeno-Associated Viruses 2 (AAV2) are modified to design therapeutic gene therapy vectors, but how they interact with the guardians of host DNA remains unknown. In this work, we show that AAV2 genomes compete with the host cell for the single-stranded DNA binding protein RPA, rendering the host vulnerable to replication stress leading to both suppression of the viral gene expression and induction of cellular DNA breaks. These findings provide insights into how gene therapies delivered at high doses could have genotoxic effects, underscoring the importance of engineering AAV2-based gene therapy platforms that express efficiently at lower doses.

microbiology↗

bioRxiv2025

IFIT3 RNA-binding activity promotes influenza A virus infection and translation efficiency

Host cells produce a vast network of antiviral factors in response to viral infection. The interferon-induced proteins with tetratricopeptide repeats (IFITs) are important effectors of a broad-spectrum antiviral response. In contrast to their canonical roles, we previously identified IFIT2 and IFIT3 as pro-viral host factors during influenza A virus (IAV) infection. During IAV infection, IFIT2 binds and enhances translation of AU-rich cellular mRNAs, including many IFN-simulated gene products, establishing a model for its broad antiviral activity. But, IFIT2 also bound viral mRNAs and enhanced their translation resulting in increased viral replication. The ability of IFIT3 to bind RNA and whether this is important for its function was not known. Here we validate direct interactions between IFIT3 and RNA using electromobility shift assays (EMSAs). RNA-binding site identification (RBS-ID) experiments then identified an RNA-binding surface composed of residues conserved in IFIT3 orthologs and IFIT2 paralogs. Mutation of the RNA-binding site reduced the ability IFIT3 to promote IAV gene expression and translation efficiency when compared to wild type IFIT3. The functional units of IFIT2 and IFIT3 are homo- and heterodimers, however the RNA-binding surfaces are located near the dimerization interface. Using co-immunoprecipitation, we showed that mutations to these sites do not affect dimerization. Together, these data establish the link between IFIT3 RNA-binding and its ability to modulate translation of host and viral mRNAs during IAV infection. ImportanceInfluenza A viruses (IAV) cause considerable morbidity and mortality through sporadic pandemics as well as annual epidemics. Zoonotic IAV strains pose an additional risk of spillover into a naive human population where prior immunity can have minimal effect. In this case, the first line of defense in the host is the innate immune response. Interferon stimulated genes (ISGs) produce a suite of proteins that are front-line effectors of innate immune responses. While ISGs are typically considered antiviral, new work has revealed an emerging trend where viruses co-opt ISGs for pro-viral function. Here, we determine how the ISG IFIT3 is used by IAV as a pro-viral factor, advancing our understanding of IFIT3 function generally as well as specifically in the context of IAV infection.

microbiology↗

bioRxiv2025

Thermodynamics shape the in vivo enzyme burden of glycolytic pathways

Thermodynamically constrained reactions and pathways are hypothesized to impose greater protein demands on cells, requiring higher enzyme amounts to sustain a given flux compared to those with stronger thermodynamics. To test this, we quantified the absolute concentrations of glycolytic enzymes in three bacterial species --Zymomonas mobilis, Escherichia coli, and Clostridium thermocellum-- which employ distinct glycolytic pathways with varying thermodynamic driving forces. By integrating enzyme concentration data with corresponding in vivo metabolic fluxes and{Delta} G measurements, we found that the highly favorable Entner-Doudoroff (ED) pathway in Z. mobilis requires only one-fourth the amount of enzymatic protein to sustain the same flux as the thermodynamically constrained pyrophosphate-dependent glycolytic pathway in C. thermocellum, with the Embden-Meyerhof-Parnas (EMP) pathway in E. coli exhibiting intermediate thermodynamic favorability and enzyme demand. Across all three pathways, early reactions with stronger thermodynamic driving forces generally required lower enzyme investment than later, less favorable steps. Additionally, reflecting differences in glycolytic strategies, the highly reversible ethanol fermentation pathway in C. thermocellum requires 10-fold more protein to maintain the same flux as the irreversible, forward-driven ethanol fermentation pathway in Z. mobilis. Thus, thermodynamic driving forces constitute a major in vivo determinant of the enzyme burden in metabolic pathways.

systems biology↗

bioRxiv2025

A Technical Evaluation of Plasma Proteomics Technologies

Plasma proteomic technologies are rapidly evolving and of critical importance to the field of biomedical research. Here we report a technical evaluation of six notable plasma proteomic technologies - unenriched (Neat), Acid depletion, PreOmics ENRICHplus, Mag-Net, Seer Proteograph XT, Olink Explore HT. The methods were compared on proteomic depth, reproducibility, linearity, tolerance to lipid interference, and limit of detection/quantification. In total we performed 618 LC-MS/MS experiments and 93 Olink Explore HT assays. The Seer method achieved the greatest proteomic depth ([~]4,500), while Olink detected [~]2,600 proteins. Other MS-based methods ranged from [~]500-2,200. Neat, Mag-Net, Seer, and Olink had strong reproducibility, while PreOmics and Acid showed higher variability. All MS methods showed good linearity with spiked-in C-Reactive Protein (CRP); CRP was surprisingly not in the Olink assay. None of the methods were affected by lipid interference. Seer had more than double the number of quantifiable proteins (4,800) for both LOD and LOQ than the next best method. Olink was comparable to Neat and Mag-Net for LOD, but worse for LOQ. Finally, we tested the applicability of these methods for detecting differences between healthy and cancer groups in a non-small cell lung cancer (NSCLC) cohort.

systems biology↗

bioRxiv2025

pH adjustment increases biofuel production from inhibitory switchgrass hydrolysates

Biofuels derived from renewable and sustainable lignocellulosic biomass, such as switchgrass, offer a promising means to limit greenhouse gas emissions. However, switchgrass grown under drought conditions contains high levels of chemical compounds that inhibit microbial conversion to biofuels. Fermentation of drought switchgrass hydrolysates by engineered Saccharomyces cerevisiae and Zymomonas mobilis generates less ethanol than fermentation of hydrolyzed switchgrass from an average rainfall year. Here, we demonstrate that this inhibitory effect can be alleviated by altering the pH of drought-switchgrass hydrolysates made from two different pretreatment methods: Ammonia Fiber Expansion (AFEX) and Soaking in Aqueous Ammonia (SAA). Fermentation rates and biofuel production from AFEX- and SAA-pretreated switchgrass hydrolysates from normal and drought years were higher at pH 5.8 than at pH 5.0 for both Saccharomyces cerevisiae and Zymomonas mobilis. Additionally, SAA pretreatment of drought switchgrass enabled increased fermentation rates and titers compared to AFEX pretreatment. Using a synthetic mimic of switchgrass hydrolysate, we identified relief from pH-dependent inhibition by lignocellulose-derived inhibitors as the cause of increased biofuel production above a pH of 5.0. These results demonstrate that SAA pretreatment and pH adjustment can significantly improve fermentation and biofuel production from switchgrass hydrolysates and especially from drought-switchgrass hydrolysates by industrial microorganisms.

microbiology↗

bioRxiv2024

SPE-CZE-MS quantifies zeptomole concentrations ofphosphorylated peptides

Capillary zone electrophoresis (CZE) is gaining attention in the field of single-cell proteomics for its ultra-low-flow and high-resolution separation abilities. Even more sample-limited yet rich in biological information are phosphoproteomics experiments, as the phosphoproteome composes only a fraction of the whole cellular proteome. Rapid analysis, high sensitivity, and maximization of sample utilization are paramount for single-cell analysis. Some challenges of coupling CZE analysis with mass spectrometry analysis (MS) of complex mixtures include 1. sensitivity due to volume loading limitations of CZE and 2. incompatibility of MS duty cycles with electrophoretic timescales. Here, we address these two challenges as applied to single-cell equivalent phosphoproteomics experiments by interfacing a microchip-based CZE device integrated with a solid-phase-extraction (SPE) bed with the Orbitrap Astral mass spectrometer. Using 225 phosphorylated peptide standards and phosphorylated peptide-enriched mouse brain tissue, we investigate microchip-based SPE-CZE functionality, quantitative performance, and complementarity to nano-LC-MS (nLC-MS) analysis. We highlight unique SPE-CZE separation mechanisms that can empower fit-for-purpose applications in single-cell-equivalent phosphoproteomics.

systems biology↗

bioRxiv2024

Deep Learning Predicts Non-Normal Peptide FAIMS Mobility Distributions Directly from Sequence

Peptide ion mobility adds an extra dimension of separation to mass spectrometry-based proteomics. The ability to accurately predict peptide ion mobility would be useful to expedite assay development and to discriminate true answers in database search. There are methods to accurately predict peptide ion mobility through drift tube devices, but methods to predict mobility through high-field asymmetric waveform ion mobility (FAIMS) are underexplored. Here, we successfully model peptide ions FAIMS mobility using a multi-label multi-output classification scheme to account for non-normal transmission distributions. We trained two models from over 100,000 human peptide precursors: a random forest and a long-term short-term memory (LSTM) neural network. Both models had different strengths, and the ensemble average of model predictions produced higher F2 score than either model alone. Finally, we explore cases where the models make mistakes and demonstrate predictive performance of F2=0.66 (AUROC=0.928) on a new test dataset of nearly 40,000 different E. coli peptide ions. The deep learning model is easily accessible via https://faims.xods.org.

bioinformatics↗

bioRxiv2024

Altering translation allows E. coli to overcome chemically stabilized G-quadruplexes

G-quadruplex (G4) structures can form in guanine-rich DNA or RNA and have been found to modulate cellular processes including replication, transcription, and translation. Many studies on the cellular roles of G4s have focused on eukaryotic systems, with far fewer probing bacterial G4s. Using a chemical-genetic approach, we identified genes in Escherichia coli that are important for growth in G4-stabilizing conditions. Reducing levels of elongation factor Tu or slowing translation elongation with chloramphenicol suppress the effects of G4 stabilization. In contrast, reducing expression of certain translation termination or ribosome recycling proteins is detrimental to growth in G4-stabilizing conditions. Proteomic and transcriptomic analyses demonstrate that ribosome assembly factors and other proteins involved in translation are less abundant in G4-stabilizing conditions. Our integrated systems approach allowed us to propose a model for how RNA G4s can present barriers to E. coli growth and that reducing the rate of translation can compensate for G4-related stress.

biochemistry↗

bioRxiv2024

Substrate-directed control of N-glycosylation in the endoplasmic reticulum calibrates signal reception at the cell-surface

One-fifth of human proteins are N-glycosylated in the endoplasmic reticulum (ER) by two oligosaccharyltransferases, OST-A and OST-B. Contrary to the prevailing view of N-glycosylation as a housekeeping function, we identified an ER pathway that modulates the activity of OST-A. Genetic analyses linked OST-A to HSP90B1, an ER chaperone for membrane receptors, and CCCD134, an ER protein we identify as the first specificity factor for N-glycosylation. During its translocation into the ER, a N-terminal peptide in HSP90B1 functions as a pseudosubstrate inhibitor of OST-A and templates the assembly of specialized ER translocon complexes containing CCDC134. Unexpectedly, OST-A functions as a scaffold rather than an enzyme in this context, stabilizing HSP90B1 by preventing its hyperglycosylation and degradation. Disruption of this pathway impairs WNT signaling at the cell surface and causes the bone developmental disorder Osteogenesis Imperfecta. Thus, N-glycosylation can be regulated by ER factors to control cell-surface receptor signaling and tissue development. One-Sentence SummaryN-glycosylation of asparagine residues on proteins can be regulated by specificity factors in the endoplasmic reticulum to control cell-surface signaling and tissue development.

cell biology↗

bioRxiv2024

Lysosomal storage disease proteo/lipidomic profiling using nMOST links ferritinophagy with mitochondrial iron deficiencies in cells lacking NPC2

Lysosomal storage diseases (LSDs) comprise [~]50 monogenic disorders marked by the buildup of cellular material in lysosomes, yet systematic global molecular phenotyping of proteins and lipids is lacking. We present a nanoflow-based multi-omic single-shot technology (nMOST) workflow that quantifies HeLa cell proteomes and lipidomes from over two dozen LSD mutants. Global cross-correlation analysis between lipids and proteins identified autophagy defects, notably the accumulation of ferritinophagy substrates and receptors, especially in NPC1-/- and NPC2-/- mutants, where lysosomes accumulate cholesterol. Autophagic and endocytic cargo delivery failures correlated with elevated lyso-phosphatidylcholine species and multi-lamellar structures visualized by cryo-electron tomography. Loss of mitochondrial cristae, MICOS- complex components, and OXPHOS components rich in iron-sulfur cluster proteins in NPC2-/- cells was largely alleviated when iron was provided through the transferrin system. This study reveals how lysosomal dysfunction affects mitochondrial homeostasis and underscores nMOST as a valuable discovery tool for identifying molecular phenotypes across LSDs.

cell biology↗

bioRxiv2023

One-Hour Phosphoproteome Analysis with the Orbitrap Astral Mass Spectrometer

Owing to its roles in cellular signal transduction, protein phosphorylation plays critical roles in myriad cell processes. That said, detecting and quantifying protein phosphorylation has remained a challenge. We describe the use of a novel mass spectrometer (Orbitrap Astral) coupled with data-independent acquisition (DIA) to achieve rapid and deep analysis of human and mouse phosphoproteomes. With this method we map approximately 30,000 unique human phosphorylation sites within a half-hour of data collection. The technology was benchmarked to other state-of-the-art MS platforms using both synthetic peptide standards and with EGF-stimulated HeLa cells. We applied this approach to generate a phosphoproteome multi-tissue atlas of the mouse. Altogether, we detected 81,120 unique phosphorylation sites within 12 hours of measurement. With this unique dataset, we examine the sequence, structural, and kinase specificity context of protein phosphorylation. Finally, we highlight the discovery potential of this resource with multiple examples of novel phosphorylation events relevant to mitochondrial and brain biology.

systems biology↗