Cytokines in the grass, a lesson learnt: Measuring cytokines in plasma using multiple reaction monitoring mass spectrometry.

RATIONALE: Cytokines are cell regulatory molecules of high importance as indicators for homeostasis and pathology in many species. The current method to measure cytokines in body fluids is reagent dependent, requiring highly specific paired antibodies. METHODS: A liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS)-based approach was developed to simultaneously establish the limits of detection (LODs) and quantification (LOQs) for recombinant cytokines IL-1ß, IL-6, IFNγ and TNFα as pure standards and in bovine sera. All experimental LC/MRM-MS data are available at CSIRO Data Access Portal repository under identifier doi.org/10.25919/5de8a0232a862. RESULTS: The present method enabled LODs and LOQs as low as 1.05 and 1.12 fmol/µL in the experiment comprised of pure standards. Comparable results were obtained in the experiment where digested cytokines were mixed with pre-digested sera proteins. The intrinsic matrix effects were evident when intact cytokines were co-digested within undiluted and undigested sera decreasing the ability to detect and quantify cytokines by 10,000-fold compared with pure standards and pre-digested sera. CONCLUSIONS: The developed LC/MRM-MS method provided insights into the difficulties in detecting the target peptides when embedded in complex matrices. Nonetheless, the method may potentially be readily applied in biomarker-focused research interrogating fluids of lesser complexity such as synovial fluid, cerebrospinal fluid and tissue culture media.

Integrative Proteomic Analysis of Digestive Tract Glycosidases from the Invasive Golden Apple Snail, Pomacea canaliculata.

The freshwater snail Pomacea canaliculata, an invasive species of global significance, possesses a well-developed digestive system and diverse feeding mechanisms enabling the intake of a wide variety of food. The identification of glycosidases in adult snails would increase the understanding of their digestive physiology and potentially generate new opportunities to eradicate and/or control this invasive species. In this study, liquid chromatography coupled to tandem mass spectrometry was applied to define the occurrence, diversity, and origin of glycoside hydrolases along the digestive tract of P. canaliculata. A range of cellulases, hemicellulases, amylases, maltases, fucosidases, and galactosidases were identified across the digestive tract. The digestive gland and the contents of the crop and style sac yield a higher diversity of glycosidase-derived peptides. Subsequently, peptides derived from 81 glycosidases (46 proteins from the public database and 35 uniquely from the transcriptome database) that were distributed among 13 glycoside hydrolase families were selected and quantified using multiple reaction monitoring mass spectrometry. This study showed a high glycosidase abundance and diversity in the gut contents of P. canaliculata which participate in extracellular digestion of complex dietary carbohydrates. Salivary and digestive glands were the main tissues involved in their synthesis and secretion.

Proteomic analysis of digestive tract peptidases and lipases from the invasive gastropod Pomacea canaliculata.

BACKGROUND: The invasive gastropod Pomacea canaliculata has received great attention in the last decades as a result of its negative impact on crops agriculture, yet knowledge of their digestive physiology remains incomplete, particularly the enzymatic breakdown of macromolecules such as proteins and lipids. RESULTS: Discovery proteomics revealed aspartic peptidases, cysteine peptidases, serine peptidases, metallopeptidases and threonine peptidases, as well as acid and neutral lipases and phospholipases along the digestive tract of P. canaliculata. Peptides specific to peptidases (139) and lipases (14) were quantified by targeted mass spectrometry. Digestion begins in the mouth via diverse salivary peptidases (nine serine peptidases; seven cysteine peptidases, one aspartic peptidase and 22 metallopeptidases) and then continues in the oesophagus (crop) via three luminal metallopeptidases (Family M12) and six serine peptidases (Family S1). Downstream, the digestive gland provides a battery of enzymes composed of aspartic peptidase (one), cysteine peptidases (nine), serine peptidases (12) and metallopeptidases (24), including aminopeptidases, carboxypeptidases and dipeptidases). The coiled gut has M1 metallopeptidases that complete the digestion of small peptides. Lipid extracellular digestion is completed by triglyceride lipases. CONCLUSION: From an integrative physiological and anatomical perspective, P. canaliculata shows an unexpected abundance and diversity of peptidases, which participate mainly in extracellular digestion. Moreover, the previously unknown occurrence of luminal lipases from the digestive gland is reported for the first time. Salivary and digestive glands were the main tissues involved in the synthesis and secretion of these enzymes, but plausibly the few luminally exclusive peptidases are secreted by ventrolateral pouches or epithelial unicellular glands. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Post-prandial response in hepatopancreas and haemolymph of Penaeus monodon fed different diets. Omics insights into glycoconjugate metabolism, energy utilisation, chitin biosynthesis, immune function, and autophagy.

Raw materials or bioactive ingredients trigger mechanisms to assimilate nutrients and activate metabolic pathways that promote growth, immune function, or energy storage. Our understanding of these processes at a molecular level remains limited in aquaculture, especially in shrimp. Here, hepatopancreas proteomics and haemolymph metabolomics were used to investigate the post-prandial response of black tiger shrimps (Penaeus monodon) fed a conventional fishmeal diet (FM); a diet supplemented with the microbial biomass Novacq™ (NV); krill meal (KM); or, fasted (FS). Using FM as a control, a 2-fold change in abundance threshold was implemented to determine the significance of proteins and metabolites. NV fed shrimp showed preference for energy derived from carbohydrates indicated by a strong signature of glycoconjugate metabolism and activation of the amino- and nucleotide sugar metabolic pathway. KM activated the glyoxylate and dicarboxylate pathway that denoted shrimp preference for lipidic energy. KM also influenced energy generation by the TCA cycle inferred from higher abundance of the metabolites succinic semialdehyde, citric acid, isocitrate, alpha ketoglutarate and ATP and downregulation of the enzyme isocitrate dehydrogenase that catalyses oxidative decarboxylation of isocitrate. FS shrimp displayed down-regulation of oxidative phosphorylation and resorted to internal lipid reserves for energy homeostasis displaying a strong signature of autophagy. Pyrimidine metabolism was the preferred energy strategy in this group. Our study also provided evidence that during fasting or consumption of specific ingredients, shrimp share common pathways to meet their energy requirements, however, the intensity at which these pathways were impacted was diet dependent.

Biochemical metabolomic profiling of the Crown-of-Thorns Starfish (Acanthaster): New insight into its biology for improved pest management.

The Crown-of-Thorns Starfish (COTS), Acanthaster species, is a voracious coral predator that destroys coral reefs when in outbreak status. The baseline metabolite and lipid biomolecules of 10 COTS tissues, including eggs from gravid females, were investigated in this study to provide insight into their biology and identify avenues for control. Targeted and untargeted metabolite- and lipidomics-based mass spectrometry approaches were used to obtain tissue-specific metabolite and lipid profiles. Across all COTS tissues, 410 metabolites and 367 lipids were identified. Most abundant were amino acids and peptides (18.7%) and wax esters (17%). There were 262 metabolites and 192 lipids identified in COTS eggs. Wax esters were more abundant in the eggs (30%) followed by triacylglycerols (TG), amino acids, and peptides. The diversity of asterosaponins in eggs (34) was higher than in tissues (2). Several asterosaponins known to modulate sperm acrosome reaction were putatively identified, including glycoside B, asterosaponin-4 (Co-Aris III), and regularoside B (asterosaponin A). The saponins saponin A, thornasteroside A, hillaside B, and non-saponins dictyol J and axinellamine B which have been shown to possess defensive properties, were found in abundance in gonads, skin, and radial nerve tissues. Inosine and 2-hexyldecanoic acid are the most abundant metabolites in tissues and eggs. As a secondary metabolite of purine degradation, inosine plays an important role in purine biosynthesis, while 2-hexyldecanoic acid is known to suppress side-chain crystallization during the synthesis of amphiphilic macromolecules (i.e., phospholipids). These significant spatial changes in metabolite, lipid, and asterosaponin profiles enabled unique insights into key biological tissue-specific processes that could be manipulated to better control COTS populations. Our findings highlight COTS as a novel source of molecules with therapeutic and cosmetic properties (ceramides, sphingolipids, carnosine, and inosine). These outcomes will be highly relevant for the development of strategies for COTS management including chemotaxis-based biocontrol and exploitation of COTS carcasses for the extraction of commercial molecules.

Multi-omics study identifies novel signatures of DNA/RNA, amino acid, peptide, and lipid metabolism by simulated diabetes on coronary endothelial cells.

Coronary artery endothelial cells (CAEC) exert an important role in the development of cardiovascular disease. Dysfunction of CAEC is associated with cardiovascular disease in subjects with type 2 diabetes mellitus (T2DM). However, comprehensive studies of the effects that a diabetic environment exerts on this cellular type are scarce. The present study characterized the molecular perturbations occurring on cultured bovine CAEC subjected to a prolonged diabetic environment (high glucose and high insulin). Changes at the metabolite and peptide level were assessed by Liquid Chromatography-Mass Spectrometry (LC-MS2) and chemoinformatics. The results were integrated with published LC-MS2-based quantitative proteomics on the same in vitro model. Our findings were consistent with reports on other endothelial cell types and identified novel signatures of DNA/RNA, amino acid, peptide, and lipid metabolism in cells under a diabetic environment. Manual data inspection revealed disturbances on tryptophan catabolism and biosynthesis of phenylalanine-based, glutathione-based, and proline-based peptide metabolites. Fluorescence microscopy detected an increase in binucleation in cells under treatment that also occurred when human CAEC were used. This multi-omics study identified particular molecular perturbations in an induced diabetic environment that could help unravel the mechanisms underlying the development of cardiovascular disease in subjects with T2DM.

Resolving hemocyanin isoform complexity in haemolymph of black tiger shrimp Penaeus monodon - implications in aquaculture, medicine and food safety.

Hemocyanin (Hc) is a multifunctional macromolecule involved in oxygen transport and non-specific immunity in shrimp. Hc is crucial in physiology and nutrition linked with optimal performance in aquaculture production systems. In medicine, Hc has been approved for clinical use in humans as adjuvant and anticancer therapeutic. In contrast, Hc has also been identified as one of the proteins causing anaphylaxis following shrimp consumption. The role of individual Hc isoforms remains unknown due to a lack of resolved Hc isoforms. We successfully identified eleven different Penaeus monodon hemocyanin (PmoHc) γ isoforms including two truncated isoforms (50 and 20 kDa) and one PmoHc ß isoform in haemolymph using proteomics informed by transcriptomics. Amino acid sequence homology ranged from 24 to 97% between putative PmoHc gene isoforms. Hc isoforms showed specific patterns of transcript expression in shrimp larval stages and adult hepatopancreas. These findings enable isoform level investigations aiming to define molecular mechanisms underpinning Hc functionality in shrimp physiology and immunity, as well as their individual immunogenic role in human allergy. Our research demonstrates the power of proteomics informed by transcriptomics to resolve isoform complexity in non-model organisms and lay the foundations for improved performance within the aquaculture industry and advance allergenic applications in medicine. SIGNIFICANCE: The roles of hemocyanin (Hc) in shrimp homeostasis and immunity as well as in human allergy are not well understood because the complexity of Hc isoforms has remained unresolved. Our results have confirmed the existence of at least 12 individual Hc isoforms in shrimp haemolymph and validated putative Hc gene assemblies from transcriptomics. Our findings will enable monitoring the expression of specific Hc isoforms in shrimp haemolymph during different environmental, nutritional and pathogenic conditions, thus providing insights into isoform specific functional roles. In medicine, the potential allergenicity of each Hc isoform could be determined and advance allergenic applications. Lastly, since Hc comprises up to 95% of the total protein in haemolymph, these isoforms become ideal targets for prawn provenance, traceability and food contamination studies.

Chemical Profiling Provides Insights into the Metabolic Machinery of Hydrocarbon-Degrading Deep-Sea Microbes.

Marine microbes are known to degrade hydrocarbons; however, microbes inhabiting deep-sea sediments remain largely unexplored. Previous studies into the classical pathways of marine microbial metabolism reveal diverse chemistries; however, metabolic profiling of marine microbes cultured with hydrocarbons is limited. In this study, taxonomic (amplicon sequencing) profiles of two environmental deep-sea sediments (>1,200 m deep) were obtained, along with taxonomic and metabolomic (mass spectrometry-based metabolomics) profiles of microbes harbored in deep-sea sediments cultured with hydrocarbons as the sole energy source. Samples were collected from the Gulf of México (GM) and cultured for 28 days using simple (toluene, benzene, hexadecane, and naphthalene) and complex (petroleum API 40) hydrocarbon mixtures as the sole energy sources. The sediment samples harbored diverse microbial communities predominantly classified into Woeseiaceae and Kiloniellaceae families, whereas Pseudomonadaceae and Enterobacteriaceae families prevailed after sediments were cultured with hydrocarbons. Chemical profiling of microbial metabolomes revealed diverse chemical groups belonging primarily to the lipids and lipid-like molecules superclass, as well as the organoheterocyclic compound superclass (ClassyFire annotation). Metabolomic data and prediction of functional profiles indicated an increase in aromatic and alkane degradation in samples cultured with hydrocarbons. Previously unreported metabolites, identified as intermediates in the degradation of hydrocarbons, were annotated as hydroxylated polyunsaturated fatty acids and carboxylated benzene derivatives. In summary, this study used mass spectrometry-based metabolomics coupled to chemoinformatics to demonstrate how microbes from deep-sea sediments could be cultured in the presence of hydrocarbons. This study also highlights how this experimental approach can be used to increase the understanding of hydrocarbon degradation by deep-sea sediment microbes.IMPORTANCE High-throughput technologies and emerging informatics tools have significantly advanced knowledge of hydrocarbon metabolism by marine microbes. However, research into microbes inhabiting deep-sea sediments (>1,000 m) is limited compared to those found in shallow waters. In this study, a nontargeted and nonclassical approach was used to examine the diversity of bacterial taxa and the metabolic profiles of hydrocarbon-degrading deep-sea microbes. In conclusion, this study used metabolomics and chemoinformatics to demonstrate that microbes from deep-sea sediment origin thrive in the presence of toxic and difficult-to-metabolize hydrocarbons. Notably, this study provides evidence of previously unreported metabolites and the global chemical repertoire associated with the metabolism of hydrocarbons by deep-sea microbes.

Using LC-MS to examine the fermented food products vinegar and soy sauce for the presence of gluten.

A strict, lifelong gluten-free (GF) diet is currently the only treatment for coeliac disease (CD). Vinegar and soy sauce are fermented condiments that often include wheat and/or barley. During fermentation cereal proteins are partially degraded by enzymes to yield peptide fragments and amino acids. Whether these fermented products contain intact or degraded gluten proteins and if they are safe for people with CD remains in question. LC-MS offers the benefit of being able to detect hydrolysed gluten that might be present in commercial vinegar and soy sauce products. LC-MS revealed the presence of gluten in malt vinegar, wherein the identified peptides derived from B-, D- and γ-hordein from barley, as well as γ-gliadin, and HMW- and LMW-glutenins from wheat that are known to contain immunopathogenic epitopes. No gluten was detected in the soy sauces examined despite wheat being a labelled ingredient indicating extensive hydrolysis of gluten during soy sauce production.

Identification of differentially expressed reproductive and metabolic proteins in the female abalone (Haliotis laevigata) gonad following artificial induction of spawning.

Inefficient control of temperate abalone spawning prevents pair-wise breeding and production of abalone with highly marketable traits. Traditionally, abalone farmers have used a combination of UV irradiation and application of temperature gradients to the tank water to artificially induce spawning. Proteins are known to regulate crucial processes such as respiration, muscle contraction, feeding, growth and reproduction. Spawning as a pre-requisite of abalone reproduction is likely to be regulated, in part, by endogenous proteins. A first step in elucidating the mechanisms that regulate spawning is to identify which proteins are directly involved during spawning. The present study examined protein expression following traditional spawning induction in the Haliotis laevigata female. Gonads were collected from abalone in the following physiological states: (1) spawning; (2) post-spawning; and (3) failed-to-spawn. Differential protein abundance was initially assessed using two-dimensional difference in-gel electrophoresis coupled with mass spectrometry for protein identification. A number of reproductive proteins such as vitellogenin, vitelline envelope zona pellucida domain 29 and prohibitin, and metabolic proteins such as thioredoxin peroxidase, superoxide dismutase and heat shock proteins were identified. Differences in protein abundance levels between physiological states were further assessed using scheduled multiple reaction monitoring mass spectrometry. Positive associations were observed between the abundance of specific proteins, such as heat shock cognate 70 and peroxiredoxin 6, and the propensity or failure to spawn in abalone. These findings have contributed to better understand both the effects of oxidative and heat stress over abalone physiology and their influence on abalone spawning.

Exploiting genomic data to identify proteins involved in abalone reproduction.

Aside from their critical role in reproduction, abalone gonads serve as an indicator of sexual maturity and energy balance, two key considerations for effective abalone culture. Temperate abalone farmers face issues with tank restocking with highly marketable abalone owing to inefficient spawning induction methods. The identification of key proteins in sexually mature abalone will serve as the foundation for a greater understanding of reproductive biology. Addressing this knowledge gap is the first step towards improving abalone aquaculture methods. Proteomic profiling of female and male gonads of greenlip abalone, Haliotis laevigata, was undertaken using liquid chromatography-mass spectrometry. Owing to the incomplete nature of abalone protein databases, in addition to searching against two publicly available databases, a custom database comprising genomic data was used. Overall, 162 and 110 proteins were identified in females and males respectively with 40 proteins common to both sexes. For proteins involved in sexual maturation, sperm and egg structure, motility, acrosomal reaction and fertilization, 23 were identified only in females, 18 only in males and 6 were common. Gene ontology analysis revealed clear differences between the female and male protein profiles reflecting a higher rate of protein synthesis in the ovary and higher metabolic activity in the testis. BIOLOGICAL SIGNIFICANCE: A comprehensive mass spectrometry-based analysis was performed to profile the abalone gonad proteome providing the foundation for future studies of reproduction in abalone. Key proteins involved in both reproduction and energy balance were identified. Genomic resources were utilised to build a database of molluscan proteins yielding >60% more protein identifications than in a standard workflow employing public protein databases.