High expression of oleoyl-ACP hydrolase underpins life-threatening respiratory viral diseases.

Respiratory infections cause significant morbidity and mortality, yet it is unclear why some individuals succumb to severe disease. In patients hospitalized with avian A(H7N9) influenza, we investigated early drivers underpinning fatal disease. Transcriptomics strongly linked oleoyl-acyl-carrier-protein (ACP) hydrolase (OLAH), an enzyme mediating fatty acid production, with fatal A(H7N9) early after hospital admission, persisting until death. Recovered patients had low OLAH expression throughout hospitalization. High OLAH levels were also detected in patients hospitalized with life-threatening seasonal influenza, COVID-19, respiratory syncytial virus (RSV), and multisystem inflammatory syndrome in children (MIS-C) but not during mild disease. In olah-/- mice, lethal influenza infection led to survival and mild disease as well as reduced lung viral loads, tissue damage, infection-driven pulmonary cell infiltration, and inflammation. This was underpinned by differential lipid droplet dynamics as well as reduced viral replication and virus-induced inflammation in macrophages. Supplementation of oleic acid, the main product of OLAH, increased influenza replication in macrophages and their inflammatory potential. Our findings define how the expression of OLAH drives life-threatening viral disease.

N-Terminomic Changes in Neurons During Excitotoxicity Reveal Proteolytic Events Associated With Synaptic Dysfunctions and Potential Targets for Neuroprotection.

Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by the overstimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIß (CaMKIIß). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbing synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src, protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIß, undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, by identifying the neuronal proteins undergoing proteolysis during excitotoxicity, our findings offer new insights into excitotoxic neuronal death mechanisms and reveal potential neuroprotective targets for neurological disorders.

Lipid-Dependent Activation of the Orphan G Protein-Coupled Receptor, GPR3.

The class A orphan G protein-coupled receptor (GPCR), GPR3, has been implicated in a variety of conditions, including Alzheimer's and premature ovarian failure. GPR3 constitutively couples with Gαs, resulting in the production of cAMP in cells. While tool compounds and several putative endogenous ligands have emerged for the receptor, its endogenous ligand, if it exists, remains a mystery. As novel potential drug targets, the structures of orphan GPCRs have been of increasing interest, revealing distinct modes of activation, including autoactivation, presence of constitutively activating mutations, or via cryptic ligands. Here, we present a cryo-electron microscopy (cryo-EM) structure of the orphan GPCR, GPR3 in complex with DNGαs and Gß1γ2. The structure revealed clear density for a lipid-like ligand that bound within an extended hydrophobic groove, suggesting that the observed "constitutive activity" was likely due to activation via a lipid that may be ubiquitously present. Analysis of conformational variance within the cryo-EM data set revealed twisting motions of the GPR3 transmembrane helices that appeared coordinated with changes in the lipid-like density. We propose a mechanism for the binding of a lipid to its putative orthosteric binding pocket linked to the GPR3 dynamics.

Structure activity relationship and target prediction for ABX464 analogues in Caenorhabditis elegans.

Global challenges with treatment failures and/or widespread resistance in parasitic worms against commercially available anthelmintics lend impetus to the development of new anthelmintics with novel mechanism(s) of action. The free-living nematode Caenorhabditis elegans is an important model organism used for drug discovery, including the screening and structure-activity investigation of new compounds, and target deconvolution. Previously, we conducted a whole-organism phenotypic screen of the 'Pandemic Response Box' (from Medicines for Malaria Venture, MMV) and identified a hit compound, called ABX464, with activity against C. elegans and a related, parasitic nematode, Haemonchus contortus. Here, we tested a series of 44 synthesized analogues to explore the pharmacophore of activity on C. elegans and revealed five compounds whose potency was similar or greater than that of ABX464, but which were not toxic to human hepatoma (HepG2) cells. Subsequently, we employed thermal proteome profiling (TPP), protein structure prediction and an in silico-docking algorithm to predict ABX464-target candidates. Taken together, the findings from this study contribute significantly to the early-stage drug discovery of a new nematocide based on ABX464. Future work is aimed at validating the ABX464-protein interactions identified here, and at assessing ABX464 and associated analogues against a panel of parasitic nematodes, towards developing a new anthelmintic with a mechanism of action that is distinct from any of the compounds currently-available commercially.

Thermostable allergens in canned fish: Evaluating risks for fish allergy.

BACKGROUND: Major fish allergens, including parvalbumin (PV), are heat stable and can withstand extensive cooking processes. Thus, the management of fish allergy generally relies on complete avoidance. Fish-allergic patients may be advised to consume canned fish, as some fish-allergic individuals have reported tolerance to canned fish. However, the safety of consuming canned fish has not been evaluated with comprehensive immunological and molecular analysis of canned fish products. METHODS: We characterized the in vitro immunoreactivity of serum obtained from fish-allergic subjects to canned fish. Seventeen canned fish products (salmon n = 8; tuna n = 7; sardine n = 2) were assessed for the content and integrity of PV using allergen-specific antibodies. Subsequently, the sIgE binding of five selected products was evaluated for individual fish-allergic patients (n = 53). Finally, sIgE-binding proteins were identified by mass spectrometry. RESULTS: The canned fish showed a markedly reduced PV content and binding to PV-specific antibodies compared with conventionally cooked fish. However, PV and other heat-stable fish allergens, including tropomyosin and collagen, still maintained their sIgE-binding capacity. Of 53 patients, 66% showed sIgE binding to canned fish proteins. The canned sardine contained proteins bound to sIgE from 51% of patients, followed by canned salmon (43%-45%) and tuna (8%-17%). PV was the major allergen in canned salmon and sardine. Tropomyosin and/or collagen also showed sIgE binding. CONCLUSION: We showed that canned fish products may not be safe for all fish-allergic patients. Canned fish products should only be considered into the diet of individuals with fish allergy, after detailed evaluation which may include in vitro diagnostics to various heat-stable fish allergens and food challenge conducted in suitable environments.

Phosphomatics: interactive interrogation of substrate-kinase networks in global phosphoproteomics datasets.

MOTIVATION: Mass spectrometry-based phosphoproteomics can routinely identify and quantify thousands of phosphorylated peptides from a single experiment. However interrogating possible upstream kinases and identifying key literature for phosphorylation sites is laborious and time-consuming. RESULTS: Here, we present Phosphomatics-a publicly available web resource for interrogating phosphoproteomics data. Phosphomatics allows researchers to upload phosphoproteomics data and interrogate possible relationships from a substrate-, kinase- or pathway-centric viewpoint. AVAILABILITY AND IMPLEMENTATION: Phosphomatics is freely available via the internet at: https://phosphomatics.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Influence of protein ion charge state on 213 nm top-down UVPD.

Ultraviolet photodissociation (UVPD) is a powerful and rapidly developing method in top-down proteomics. Sequence coverages can exceed those obtained with collision- and electron-induced fragmentation methods. Because of the recent interest in UVPD, factors that influence protein fragmentation and sequence coverage are actively debated in the literature. Here, we performed top-down 213 nm UVPD experiments on a 7 T Fourier-transform ion cyclotron resonance mass spectrometer (FT-ICR MS) for the model proteins ubiquitin, myoglobin and cytochrome c that were electrosprayed from native, denaturing and supercharging solutions in order to investigate the effect of protein charge states on UVPD fragments. By performing UVPD in ultrahigh vacuum, factors associated with collisional cooling and any ion activation during transfer between mass analyzers can be largely eliminated. Sequence coverage increased from <10% for low charge states to >60% for high charge states for all three proteins. This trend is influenced by the overall charge state, i.e., charges per number of amino acid residues, and to a lesser degree by associated structural changes of protein ions of different charge states based on comparisons to published collision-cross section measurements. To rationalize this finding, and correlate sequence ion formation and identity with the number and location of protons, UVPD results were compared to protonation sites predicted based on electrostatic modelling. Assuming confined protonation sites, these results indicate the presence of two general fragmentation types; i.e., charge remote and charge directed. For moderately high protein charge states, fragment ions mostly originate in regions between likely protonation sites (charge remote), whereas sequence ions of highly charge protein ions occur either near backbone amide protonation sites at low-basicity residues (charge directed) or at charge remote sites (i.e., high-basicity residues). Overall, our results suggest that top-down 213 UVPD performance in the zero-pressure limit depends strongly on protein charge states and protonation sites can influence the location of backbone cleavages.

Nontargeted Identification of Plasma Proteins O-, N-, and S-Transmethylated by O-Methyl Organophosphates.

Organophosphates (OPs) are used worldwide as pesticides. However, acute and chronic exposure to OPs can cause serious adverse health effects. The mechanism of delayed OP toxicity is thought to involve off-target inhibition of serine proteases, although the precise molecular details remain unclear owing to the lack of an analytical method for global detection of protein targets of OPs. Here, we report the development of a mass spectrometry method to identify OP-adducted proteins from complex mixtures in a nontargeted manner. Human plasma was incubated with the OP dichlorvos that was 50% isotopically labeled and 50% unlabeled. Proteins and protein adducts were extracted, digested, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect "twin ions" of peptides that were covalently modified by a chemical reaction with dichlorvos. The LC-MS/MS data were processed by a blended data analytics software (Xenophile) to detect the amino acid residue sites of proteins that were covalently modified by exposure to OPs. We discovered that OPs can transmethylate the N, S, and O side chains of His, Cys, Glu, Asp, and Lys residues. For model systems, such transmethylation reactions were confirmed by LC-MS, nuclear magnetic resonance (NMR), and rationalized using electronic structure calculations. Methylation of the ubiquitous antioxidant glutathione by dichlorvos can decrease the reducing/oxidizing equilibrium of glutathione in liver extracts, which has been implicated in diseases and pathological conditions associated with delayed OP toxicity.

Origin and Prediction of Highly Specific Bond Cleavage Sites in the Thermal Activation of Intact Protein Ions.

Predicting the fragmentation patterns of proteins would be beneficial for the reliable identification of intact proteins by mass spectrometry. However, the ability to accurately make such predictions remains elusive. An approach to predict the specific cleavage sites in whole proteins resulting from collision-induced dissociation by use of an improved electrostatic model for calculating the proton configurations of highly-charged protein ions is reported. Using ubiquitin, cytochrome c, lysozyme and ß-lactoglobulin as prototypical proteins, this approach can be used to predict the fragmentation patterns of intact proteins. For sufficiently highly charged proteins, specific cleavages occur near the first low-basicity amino acid residues that are protonated with increasing charge state. Hybrid QM/QM' (QM=quantum mechanics) and molecular dynamics (MD) simulations and energy-resolved collision-induced dissociation measurements indicated that the barrier to the specific dissociation of the protonated amide backbone bond is significantly lower than competitive charge remote fragmentation. Unlike highly charged peptides, the protons at low-basicity sites in highly charged protein ions can be confined to a limited sequence of low-basicity amino acid residues by electrostatic repulsion, which results in highly specific fragmentation near the site of protonation. This research suggests that the optimal charge states to form specific sequence ions of intact proteins in higher abundances than the use of less specific ion dissociation methods can be predicted a priori.

Nontargeted Identification of Reactive Metabolite Protein Adducts.

Metabolic bioactivation of many different chemicals results in the formation of highly reactive compounds (chemically reactive metabolites, CRMs) that can lead to toxicity via binding to macromolecular targets (e.g., proteins or DNA). There is a need to develop robust, rapid, and nontargeted analytical techniques to determine the identity of the protein targets of CRMs and their sites of modification. Here, we introduce a nontargeted methodology capable of determining both the identity of a CRM formed from an administered compound as well as the protein targets modified by the reactive metabolite in a single experiment without prior information. Acetaminophen (N-acetyl-p-aminophenol, APAP) and 13C6-APAP were incubated with rat liver microsomes, which are known to bioactivate APAP to the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI). Global tryptic digestion followed by liquid chromatographic/mass spectrometric (LC/MS) analysis was used to locate "twin" ion peaks of peptides adducted by NAPQI and for shotgun proteomics via tandem mass spectrometry (MS/MS). By the development of blended data analytics software called Xenophile, the identity of the amino acid residue that was adducted can be established, which eliminates the need for specific parametrization of protein database search algorithms. This combination of experimental design and data analysis software allows the identity of a CRM, the protein target, and the amino acid residues that are modified to be rapidly established directly from experimental data. Xenophile is freely available from https://github.com/mgleeming/Xenophile .

Highly Charged Protein Ions: The Strongest Organic Acids to Date.

The basicity of highly protonated cytochrome c (cyt c) and myoglobin (myo) ions were investigated using tandem mass spectrometry, ion-molecule reactions (IMRs), and theoretical calculations as a function of charge state. Surprisingly, highly charged protein ions (HCPI) can readily protonate non-polar molecules and inert gases, including Ar, O2 , and N2 in thermal IMRs. The most HCPIs that can be observed are over 130 kJ mol-1 less basic than the least basic neutral organic molecules known (tetrafluoromethane and methane). Based on theoretical calculations, it is predicted that protonated cyt c and myo ions should spontaneously lose a proton to vacuum for charge states in which every third residue is protonated. In this study, HCPIs are formed where every fourth residue on average is protonated. These results indicate that protein ions in higher charge states can be formed using a low-pressure ion source to reduce proton-transfer reactions between protein ions and gases from the atmosphere.

High-resolution twin-ion metabolite extraction (HiTIME) mass spectrometry: nontargeted detection of unknown drug metabolites by isotope labeling, liquid chromatography mass spectrometry, and automated high-performance computing.

The metabolic fate of a compound can often determine the success of a new drug lead. Thus, significant effort is directed toward identifying the metabolites formed from a given molecule. Here, an automated and nontargeted procedure is introduced for detecting drug metabolites without authentic metabolite standards via the use of stable isotope labeling, liquid chromatography mass spectrometry (LC/MS), and high-performance computing. LC/MS of blood plasma extracts from rats that were administered a 1:1 mixture of acetaminophen (APAP) and (13)C6-APAP resulted in mass spectra that contained "twin" ions for drug metabolites that were not detected in control spectra (i.e., no APAP administered). Because of the development of a program (high-resolution twin-ion metabolite extraction; HiTIME) that can identify twin-ions in high-resolution mass spectra without centroiding (i.e., reduction of mass spectral peaks to single data points), 9 doublets corresponding to APAP metabolites were identified. This is nearly twice that obtained by use of existing programs that make use of centroiding to reduce computational cost under these conditions with a quadrupole time-of-flight mass spectrometer. By a manual search for all reported APAP metabolite ions, no additional twin-ion signals were assigned. These data indicate that all the major metabolites of APAP and multiple low-abundance metabolites (e.g., acetaminophen hydroxy- and methoxysulfate) that are rarely reported were detected. This methodology can be used to detect drug metabolites without prior knowledge of their identity. HiTIME is freely available from https://github.com/bjpop/HiTIME .

What Are the Potential Sites of Protein Arylation by N-Acetyl-p-benzoquinone Imine (NAPQI)?

Acetaminophen (paracetamol, APAP) is a safe and widely used analgesic medication when taken at therapeutic doses. However, APAP can cause potentially fatal hepatotoxicity when taken in overdose or in patients with metabolic irregularities. The production of the electrophilic and putatively toxic compound N-acetyl-p-benzoquinone imine (NAPQI), which cannot be efficiently detoxicated at high doses, is implicated in APAP toxicity. Numerous studies have identified that excess NAPQI can form covalent linkages to the thiol side chains of cysteine residues in proteins; however, the reactivity of NAPQI toward other amino acid side chains is largely unexplored. Here, we report a survey of the reactivity of NAPQI toward 11 N-acetyl amino acid methyl esters and four peptides. (1)H NMR analysis reveals that NAPQI forms covalent bonds to the side-chain functional groups of cysteine, methionine, tyrosine, and tryptophan residues. Analogous reaction products were observed when NAPQI was reacted with synthetic model peptides GAIL-X-GAILR for X = Cys, Met, Tyr, and Trp. Tandem mass spectrometry peptide sequencing showed that the NAPQI modification sites are located on the "X" residue in each case. However, when APAP and the GAIL-X-GAILR peptide were incubated with rat liver microsomes that contain many metabolic enzymes, NAPQI formed by oxidative metabolism reacted with GAIL-C-GAILR exclusively. For the peptides where X = Met, Tyr, and Trp, competing reactions between NAPQI and alternative nucleophiles precluded arylation of the target peptide by NAPQI. Although Cys residues are favorably targeted under these conditions, these data suggest that NAPQI can, in principle, also damage proteins at Met, Tyr, and Trp residues.

Targeted-metabolomic and untargeted-proteomic approaches reveal the effects of muscle fibre type and postmortem ageing on taste-active compounds in beef.

The taste of beef is caused by taste-active compounds detected in the mouth during mastication. We hypothesised that the concentration of taste-active compounds in beef is influenced by muscle-fibre-type and postmortem ageing. To test this, and unravel the underlying mechanisms, we investigated the taste-active compounds, and proteomic profiles, in beef masseter [oxidative muscle, all type I fibres) and cutaneous trunci (glycolytic muscle, mostly type II fibres) before and after 14-days postmortem ageing. Our results showed that nucleotides were initially higher and degraded slower in cutaneous trunci (P < 0.05 for both), which could be explained by the profile of nucleotide metabolism enzymes. In contrast, free amino acids were initially higher and increased more in masseter compared to cutaneous trunci (P < 0.05 for all), which might be explained by the profile and activity of proteases in these two muscles. Our results indicate the taste of beef is affected by the muscle-fibre-type and postmortem ageing.

Physiochemical changes, metabolite discrepancies of brown seaweed-derived sulphated polysaccharides in the upper gastrointestinal tract and their effects on bioactive expression.

Brown seaweed-derived polysaccharides, notably fucoidan and laminarin, are known for their extensive array of bioactivities and physicochemical properties. However, the effects of upper digestive tract modification on the bioactive performance of fucoidan and laminarin fractions (FLFs) sourced from Australian native species are largely unknown. Here, the digestibility and bioaccessibility of FLFs were evaluated by tracking the dynamic changes in reducing sugar content (CR), profiling the free monosaccharide composition using LC-MS, and comparing high-performance gel permeation chromatography profile variation via LC-SEC-RI. The effects of digestive progression on bioactive performance were assessed by comparing the antioxidant and antidiabetic potential of FLFs and FLF digesta. We observed that molecular weight (Mw) decreased during gastric digestion indicating that FLF aggregates were disrupted in the stomach. During intestinal digestion, Mw gradually decreased and CR increased indicating cleavage of glycosidic bonds releasing free sugars. Although the antioxidant and antidiabetic capacities were not eliminated by the digestion progression, the bioactive performance of FLFs under a digestive environment was reduced contrasting with the same concentration level of the undigested FLFs. These data provide comprehensive information on the digestibility and bioaccessibility of FLFs, and shed light on the effects of digestive progression on bioactive expression.

Blood Distribution of SARS-CoV-2 Lipid Nanoparticle mRNA Vaccine in Humans.

Lipid nanoparticle mRNA vaccines are an exciting but emerging technology used in humans. There is limited understanding of the factors that influence their biodistribution and immunogenicity. Antibodies to poly(ethylene glycol) (PEG), which is on the surface of the lipid nanoparticle, are detectable in humans and boosted by human mRNA vaccination. We hypothesized that PEG-specific antibodies could increase the clearance of mRNA vaccines. To test this, we developed methods to quantify both the vaccine mRNA and ionizable lipid in frequent serial blood samples from 19 subjects receiving Moderna SPIKEVAX mRNA booster immunization. Both the vaccine mRNA and ionizable lipid peaked in blood 1-2 days post vaccination (median peak level 0.19 and 3.22 ng mL-1, respectively). The vaccine mRNA was detectable and quantifiable up to 14-15 days postvaccination in 37% of subjects. We measured the proportion of vaccine mRNA that was relatively intact in blood over time and found that the decay kinetics of the intact mRNA and ionizable lipid were identical, suggesting the intact lipid nanoparticle recirculates in blood. However, the decay rates of mRNA and ionizable lipids did not correlate with baseline levels of PEG-specific antibodies. Interestingly, the magnitude of mRNA and ionizable lipid detected in blood did correlate with the boost in the level of PEG antibodies. Furthermore, the ability of a subject's monocytes to phagocytose lipid nanoparticles was inversely related to the rise in PEG antibodies. This suggests that the circulation of mRNA lipid nanoparticles into the blood and their clearance by phagocytes influence the PEG immunogenicity of the mRNA vaccines. Overall, this work defines the pharmacokinetics of lipid nanoparticle mRNA vaccine components in human blood after intramuscular injection and the factors that influence these processes. These insights should be valuable in improving the future safety and efficacy of lipid nanoparticle mRNA vaccines and therapeutics.

Comparative structure activity and target exploration of 1,2-diphenylethynes in Haemonchus contortus and Caenorhabditis elegans.

Infections and diseases caused by parasitic nematodes have a major adverse impact on the health and productivity of animals and humans worldwide. The control of these parasites often relies heavily on the treatment with commercially available chemical compounds (anthelmintics). However, the excessive or uncontrolled use of these compounds in livestock animals has led to major challenges linked to drug resistance in nematodes. Therefore, there is a need to develop new anthelmintics with novel mechanism(s) of action. Recently, we identified a small molecule, designated UMW-9729, with nematocidal activity against the free-living model organism Caenorhabditis elegans. Here, we evaluated UMW-9729's potential as an anthelmintic in a structure-activity relationship (SAR) study in C. elegans and the highly pathogenic, blood-feeding Haemonchus contortus (barber's pole worm), and explored the compound-target relationship using thermal proteome profiling (TPP). First, we synthesised and tested 25 analogues of UMW-9729 for their nematocidal activity in both H. contortus (larvae and adults) and C. elegans (young adults), establishing a preliminary nematocidal pharmacophore for both species. We identified several compounds with marked activity against either H. contortus or C. elegans which had greater efficacy than UMW-9729, and found a significant divergence in compound bioactivity between these two nematode species. We also identified a UMW-9729 analogue, designated 25, that moderately inhibited the motility of adult female H. contortus in vitro. Subsequently, we inferred three H. contortus proteins (HCON_00134350, HCON_00021470 and HCON_00099760) and five C. elegans proteins (F30A10.9, F15B9.8, B0361.6, DNC-4 and UNC-11) that interacted directly with UMW-9729; however, no conserved protein target was shared between the two nematode species. Future work aims to extend the SAR investigation in these and other parasitic nematode species, and validate individual proteins identified here as possible targets of UMW-9729. Overall, the present study evaluates this anthelmintic candidate and highlights some challenges associated with early anthelmintic investigation.

Streamlining Protein Fractional Synthesis Rates Using SP3 Beads and Stable Isotope Mass Spectrometry: A Case Study on the Plant Ribosome.

Ribosomes are an archetypal ribonucleoprotein assembly. Due to ribosomal evolution and function, r-proteins share specific physicochemical similarities, making the riboproteome particularly suited for tailored proteome profiling methods. Moreover, the structural proteome of ribonucleoprotein assemblies reflects context-dependent functional features. Thus, characterizing the state of riboproteomes provides insights to uncover the context-dependent functionality of r-protein rearrangements, as they relate to what has been termed the ribosomal code, a concept that parallels that of the histone code, in which chromatin rearrangements influence gene expression. Compared to high-resolution ribosomal structures, omics methods lag when it comes to offering customized solutions to close the knowledge gap between structure and function that currently exists in riboproteomes. Purifying the riboproteome and subsequent shot-gun proteomics typically involves protein denaturation and digestion with proteases. The results are relative abundances of r-proteins at the ribosome population level. We have previously shown that, to gain insight into the stoichiometry of individual proteins, it is necessary to measure by proteomics bound r-proteins and normalize their intensities by the sum of r-protein abundances per ribosomal complex, i.e., 40S or 60S subunits. These calculations ensure that individual r-protein stoichiometries represent the fraction of each family/paralog relative to the complex, effectively revealing which r-proteins become substoichiometric in specific physiological scenarios. Here, we present an optimized method to profile the riboproteome of any organism as well as the synthesis rates of r-proteins determined by stable isotope-assisted mass spectrometry. Our method purifies the r-proteins in a reversibly denatured state, which offers the possibility for combined top-down and bottom-up proteomics. Our method offers a milder native denaturation of the r-proteome via a chaotropic GuHCl solution as compared with previous studies that use irreversible denaturation under highly acidic conditions to dissociate rRNA and r-proteins. As such, our method is better suited to conserve post-translational modifications (PTMs). Subsequently, our method carefully considers the amino acid composition of r-proteins to select an appropriate protease for digestion. We avoid non-specific protease cleavage by increasing the pH of our standardized r-proteome dilutions that enter the digestion pipeline and by using a digestion buffer that ensures an optimal pH for a reliable protease digestion process. Finally, we provide the R package ProtSynthesis to study the fractional synthesis rates of r-proteins. The package uses physiological parameters as input to determine peptide or protein fractional synthesis rates. Once the physiological parameters are measured, our equations allow a fair comparison between treatments that alter the biological equilibrium state of the system under study. Our equations correct peptide enrichment using enrichments in soluble amino acids, growth rates, and total protein accumulation. As a means of validation, our pipeline fails to find "false" enrichments in non-labeled samples while also filtering out proteins with multiple unique peptides that have different enrichment values, which are rare in our datasets. These two aspects reflect the accuracy of our tool. Our method offers the possibility of elucidating individual r-protein family/paralog abundances, PTM status, fractional synthesis rates, and dynamic assembly into ribosomal complexes if top-down and bottom-up proteomic approaches are used concomitantly, taking one step further into mapping the native and dynamic status of the r-proteome onto high-resolution ribosome structures. In addition, our method can be used to study the proteomes of all macromolecular assemblies that can be purified, although purification is the limiting step, and the efficacy and accuracy of the proteases may be limited depending on the digestion requirements. Key features • Efficient purification of the ribosomal proteome: streamlined procedure for the specific purification of the ribosomal proteome or complex Ome. • Accurate calculation of fractional synthesis rates: robust method for calculating fractional protein synthesis rates in macromolecular complexes under different physiological steady states. • Holistic ribosome methodology focused on plants: comprehensive approach that provides insights into the ribosomes and translational control of plants, demonstrated using cold acclimation [1]. • Tailored strategies for stable isotope labeling in plants: methodology focusing on materials and labeling considerations specific to free and proteinogenic amino acid analysis [2].

Structure-activity relationship and target investigation of 2-aryl quinolines with nematocidal activity.

Within the context of our anthelmintic discovery program, we recently identified and evaluated a quinoline derivative, called ABX464 or obefazimod, as a nematocidal candidate; synthesised a series of analogues which were assessed for activity against the free-living nematode Caenorhabditis elegans; and predicted compound-target relationships by thermal proteome profiling (TPP) and in silico docking. Here, we logically extended this work and critically evaluated the anthelmintic activity of ABX464 analogues on Haemonchus contortus (barber's pole worm) - a highly pathogenic nematode of ruminant livestock. First, we tested a series of 44 analogues on H. contortus (larvae and adults) to investigate the nematocidal pharmacophore of ABX464, and identified one compound with greater potency than the parent compound and showed moderate activity against a select number of other parasitic nematodes (including Ancylostoma, Heligmosomoides and Strongyloides species). Using TPP and in silico modelling studies, we predicted protein HCON_00074590 (a predicted aldo-keto reductase) as a target candidate for ABX464 in H. contortus. Future work aims to optimise this compound as a nematocidal candidate and investigate its pharmacokinetic properties. Overall, this study presents a first step toward the development of a new nematocide.

Simulation of mass spectrometry-based proteomics data with Synthedia.

Motivation: A large number of experimental and bioinformatic parameters must be set to identify and quantify peptides in mass spectrometry experiments and each of these will impact the results. An ability to simulate raw data with known contents would allow researchers to rapidly explore the effects of varying experimental parameters and systematically investigate downstream processing software. A range of data simulators are available for established data-dependent acquisition methodologies, but these do not extend to the rapidly developing field of data-independent acquisition (DIA) strategies. Results: Here, we present Synthedia-a software package to simulate DIA liquid chromatography-mass spectrometry for bottom-up proteomics experiments. Synthedia can generate datasets with known peptide precursor ions and fragments and allows for the customization of a wide variety of chromatographic and mass spectrometry parameters. Availability and implementation: Synthedia is freely available via the internet and can be used through a graphical website (https://synthedia.org/) or locally via the command line (https://github.com/mgleeming/synthedia/). Supplementary information: Supplementary data are available at Bioinformatics Advances online.