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.

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].

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.

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 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.

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.

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.

Integrative omics identifies conserved and pathogen-specific responses of sepsis-causing bacteria.

Even in the setting of optimal resuscitation in high-income countries severe sepsis and septic shock have a mortality of 20-40%, with antibiotic resistance dramatically increasing this mortality risk. To develop a reference dataset enabling the identification of common bacterial targets for therapeutic intervention, we applied a standardized genomic, transcriptomic, proteomic and metabolomic technological framework to multiple clinical isolates of four sepsis-causing pathogens: Escherichia coli, Klebsiella pneumoniae species complex, Staphylococcus aureus and Streptococcus pyogenes. Exposure to human serum generated a sepsis molecular signature containing global increases in fatty acid and lipid biosynthesis and metabolism, consistent with cell envelope remodelling and nutrient adaptation for osmoprotection. In addition, acquisition of cholesterol was identified across the bacterial species. This detailed reference dataset has been established as an open resource to support discovery and translational research.

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.

Thermal proteome profiling reveals Haemonchus orphan protein HCO_011565 as a target of the nematocidal small molecule UMW-868.

Parasitic roundworms (nematodes) cause destructive diseases, and immense suffering in humans and other animals around the world. The control of these parasites relies heavily on anthelmintic therapy, but treatment failures and resistance to these drugs are widespread. As efforts to develop vaccines against parasitic nematodes have been largely unsuccessful, there is an increased focus on discovering new anthelmintic entities to combat drug resistant worms. Here, we employed thermal proteome profiling (TPP) to explore hit pharmacology and to support optimisation of a hit compound (UMW-868), identified in a high-throughput whole-worm, phenotypic screen. Using advanced structural prediction and docking tools, we inferred an entirely novel, parasite-specific target (HCO_011565) of this anthelmintic small molecule in the highly pathogenic, blood-feeding barber's pole worm, and in other socioeconomically important parasitic nematodes. The "hit-to-target" workflow constructed here provides a unique prospect of accelerating the simultaneous discovery of novel anthelmintics and associated parasite-specific targets.

The Impact of Chromate on Pseudomonas aeruginosa Molybdenum Homeostasis.

Acquisition of the trace-element molybdenum via the high-affinity ATP-binding cassette permease ModABC is essential for Pseudomonas aeruginosa respiration in anaerobic and microaerophilic environments. This study determined the X-ray crystal structures of the molybdenum-recruiting solute-binding protein ModA from P. aeruginosa PAO1 in the metal-free state and bound to the group 6 metal oxyanions molybdate, tungstate, and chromate. Pseudomonas aeruginosa PAO1 ModA has a non-contiguous dual-hinged bilobal structure with a single metal-binding site positioned between the two domains. Metal binding results in a 22° relative rotation of the two lobes with the oxyanions coordinated by four residues, that contribute six hydrogen bonds, distinct from ModA orthologues that feature an additional oxyanion-binding residue. Analysis of 485 Pseudomonas ModA sequences revealed conservation of the metal-binding residues and ß-sheet structural elements, highlighting their contribution to protein structure and function. Despite the capacity of ModA to bind chromate, deletion of modA did not affect P. aeruginosa PAO1 sensitivity to chromate toxicity nor impact cellular accumulation of chromate. Exposure to sub-inhibitory concentrations of chromate broadly perturbed P. aeruginosa metal homeostasis and, unexpectedly, was associated with an increase in ModA-mediated molybdenum uptake. Elemental analyses of the proteome from anaerobically grown P. aeruginosa revealed that, despite the increase in cellular molybdenum upon chromate exposure, distribution of the metal within the proteome was substantially perturbed. This suggested that molybdoprotein cofactor acquisition may be disrupted, consistent with the potent toxicity of chromate under anaerobic conditions. Collectively, these data reveal a complex relationship between chromate toxicity, molybdenum homeostasis and anaerobic respiration.

Trace residue identification, characterization, and longitudinal monitoring of the novel synthetic opioid ß-U10, from discarded drug paraphernalia.

Empirical data regarding dynamic alterations in illicit drug supply markets in response to the COVID-19 pandemic, including the potential for introduction of novel drug substances and/or increased poly-drug combination use at the "street" level, that is, directly proximal to the point of consumption, are currently lacking. Here, a high-throughput strategy employing ambient ionization-mass spectrometry is described for the trace residue identification, characterization, and longitudinal monitoring of illicit drug substances found within >6,600 discarded drug paraphernalia (DDP) samples collected during a pilot study of an early warning system for illicit drug use in Melbourne, Australia from August 2020 to February 2021, while significant COVID-19 lockdown conditions were imposed. The utility of this approach is demonstrated for the de novo identification and structural characterization of ß-U10, a previously unreported naphthamide analog within the "U-series" of synthetic opioid drugs, including differentiation from its α-U10 isomer without need for sample preparation or chromatographic separation prior to analysis. Notably, ß-U10 was observed with 23 other drug substances, most commonly in temporally distinct clusters with heroin, etizolam, and diphenhydramine, and in a total of 182 different poly-drug combinations. Longitudinal monitoring of the number and weekly "average signal intensity" (ASI) values of identified substances, developed here as a semi-quantitative proxy indicator of changes in availability, relative purity and compositions of street level drug samples, revealed that increases in the number of identifications and ASI for ß-U10 and etizolam coincided with a 50% decrease in the number of positive detections and an order of magnitude decrease in the ASI for heroin.

Getting more out of FLAG-Tag co-immunoprecipitation mass spectrometry experiments using FAIMS.

Co-immunoprecipitation of proteins coupled to mass spectrometry is critical for the understanding of protein interaction networks. In instances where a suitable antibody is not available, it is common to graft synthetic tags onto a target protein sequence thereby allowing the use of commercially available antibodies for affinity purification. A common approach is through FLAG-Tag co-immunoprecipitation. To allow the selective elution of protein complexes, competitive displacement using a large molar excess of the tag peptides is often carried out. Yet, this creates downstream challenges for the mass spectrometry analysis due to the presence of large quantities of these peptides. Here, we demonstrate that Field Asymmetric Ion Mobility Spectrometry (FAIMS), a gas phase ion separation device prior to mass spectrometry analysis can be applied to FLAG-Tag co-immunoprecipitation experiments to increase the depth of protein coverage. By excluding these abundant tag peptides, we were able to observe deeper coverage of interacting proteins and as a result, deeper biological insights, without the need for additional sample handling or altering sample preparation protocols. SIGNIFICANCE: We have shown that application of FAIMS separation in the gas phase can increase the proteome coverage of Flag-Tagged co-immunoprecipitation mass spectrometry experiments versus one without FAIMS. We were able to observe deeper coverage of interacting proteins and as a result, deeper biological insights, without additional sample handling, fractionation, machine run time or modifying the sample preparation protocol.

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.

Membrane-Enriched Proteomics Link Ribosome Accumulation and Proteome Reprogramming With Cold Acclimation in Barley Root Meristems.

Due to their sessile nature, plants rely on root systems to mediate many biotic and abiotic cues. To overcome these challenges, the root proteome is shaped to specific responses. Proteome-wide reprogramming events are magnified in meristems due to their active protein production. Using meristems as a test system, here, we study the major rewiring that plants undergo during cold acclimation. We performed tandem mass tag-based bottom-up quantitative proteomics of two consecutive segments of barley seminal root apexes subjected to suboptimal temperatures. After comparing changes in total and ribosomal protein (RP) fraction-enriched contents with shifts in individual protein abundances, we report ribosome accumulation accompanied by an intricate translational reprogramming in the distal apex zone. Reprogramming ranges from increases in ribosome biogenesis to protein folding factors and suggests roles for cold-specific RP paralogs. Ribosome biogenesis is the largest cellular investment; thus, the vast accumulation of ribosomes and specific translation-related proteins during cold acclimation could imply a divergent ribosomal population that would lead to a proteome shift across the root. Consequently, beyond the translational reprogramming, we report a proteome rewiring. First, triggered protein accumulation includes spliceosome activity in the root tip and a ubiquitous upregulation of glutathione production and S-glutathionylation (S-GSH) assemblage machineries in both root zones. Second, triggered protein depletion includes intrinsically enriched proteins in the tip-adjacent zone, which comprise the plant immune system. In summary, ribosome and translation-related protein accumulation happens concomitantly to a proteome reprogramming in barley root meristems during cold acclimation. The cold-accumulated proteome is functionally implicated in feedbacking transcript to protein translation at both ends and could guide cold acclimation.

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.

Expanding the allergen repertoire of salmon and catfish.

BACKGROUND: Diagnostic tests for fish allergy are hampered by the large number of under-investigated fish species. Four salmon allergens are well-characterized and registered with the WHO/IUIS while no catfish allergens have been described so far. In 2008, freshwater-cultured catfish production surpassed that of salmon, the globally most-cultured marine species. We aimed to identify, quantify, and compare all IgE-binding proteins in salmon and catfish. METHODS: Seventy-seven pediatric patients with clinically confirmed fish allergy underwent skin prick tests to salmon and catfish. The allergen repertoire of raw and heated protein extracts was evaluated by immunoblotting using five allergen-specific antibodies and patients' serum followed by mass spectrometric analyses. RESULTS: Raw and heated extracts from catfish displayed a higher frequency of IgE-binding compared to those from salmon (77% vs 70% and 64% vs 53%, respectively). The major fish allergen parvalbumin demonstrated the highest IgE-binding capacity (10%-49%), followed by triosephosphate isomerase (TPI; 19%-34%) in raw and tropomyosin (6%-32%) in heated extracts. Six previously unidentified fish allergens, including TPI, were registered with the WHO/IUIS. Creatine kinase from salmon and catfish was detected by IgE from 14% and 10% of patients, respectively. Catfish L-lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, and glucose-6-phosphate isomerase showed IgE-binding for 6%-13% of patients. In salmon, these proteins could not be separated successfully. CONCLUSIONS: We detail the allergen repertoire of two highly farmed fish species. IgE-binding to fish tropomyosins and TPIs was demonstrated for the first time in a large patient cohort. Tropomyosins, in addition to parvalbumins, should be considered for urgently needed improved fish allergy diagnostics.

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.

Type IX Secretion System Cargo Proteins Are Glycosylated at the C Terminus with a Novel Linking Sugar of the Wbp/Vim Pathway.

Porphyromonas gingivalis and Tannerella forsythia use the type IX secretion system to secrete cargo proteins to the cell surface where they are anchored via glycolipids. In P. gingivalis, the glycolipid is anionic lipopolysaccharide (A-LPS), of partially known structure. Modified cargo proteins were deglycosylated using trifluoromethanesulfonic acid and digested with trypsin or proteinase K. The residual modifications were then extensively analyzed by tandem mass spectrometry. The C terminus of each cargo protein was amide-bonded to a linking sugar whose structure was deduced to be 2-N-seryl, 3-N-acetylglucuronamide in P. gingivalis and 2-N-glycyl, 3-N-acetylmannuronic acid in T. forsythia The structures indicated the involvement of the Wbp pathway to produce 2,3-di-N-acetylglucuronic acid and a WbpS amidotransferase to produce the uronamide form of this sugar in P. gingivalis The wbpS gene was identified as PGN_1234 as its deletion resulted in the inability to produce the uronamide. In addition, the P. gingivalisvimA mutant which lacks A-LPS was successfully complemented by the T. forsythiavimA gene; however, the linking sugar was altered to include glycine rather than serine. After removal of the acetyl group at C-2 by the putative deacetylase, VimE, VimA presumably transfers the amino acid to complete the biosynthesis. The data explain all the enzyme activities required for the biosynthesis of the linking sugar accounting for six A-LPS-specific genes. The linking sugar is therefore the key compound that enables the attachment of cargo proteins in P. gingivalis and T. forsythia We propose to designate this novel linking sugar biosynthetic pathway the Wbp/Vim pathway.IMPORTANCEPorphyromonas gingivalis and Tannerella forsythia, two pathogens associated with severe gum disease, use the type IX secretion system (T9SS) to secrete and attach toxic arrays of virulence factor proteins to their cell surfaces. The proteins are tethered to the outer membrane via glycolipid anchors that have remained unidentified for more than 2 decades. In this study, the first sugar molecules (linking sugars) in these anchors are identified and found to be novel compounds. The novel biosynthetic pathway of these linking sugars is also elucidated. A diverse range of bacteria that do not have the T9SS were found to have the genes for this pathway, suggesting that they may synthesize similar linking sugars for utilization in different systems. Since the cell surface attachment of virulence factors is essential for virulence, these findings reveal new targets for the development of novel therapies.