Mass Spectrometry-based Untargeted Metabolomics Research Articles

Leech Poecilobdella manillensis protein extract ameliorated hyperuricemia by restoring gut microbiota dysregulation and affecting serum metabolites

BACKGROUND Hyperuricemia (HUA) is a public health concern that needs to be solved urgently. The lyophilized powder of Poecilobdella manillensis has been shown to significantly alleviate HUA; however, its underlying metabolic regulation remains unclear. AIM To explore the underlying mechanisms of Poecilobdella manillensis in HUA based on modulation of the gut microbiota and host metabolism. METHODS A mouse model of rapid HUA was established using a high-purine diet and potassium oxonate injections. The mice received oral drugs or saline. Additionally, 16S rRNA sequencing and ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry-based untargeted metabolomics were performed to identify changes in the microbiome and host metabolome, respectively. The levels of uric acid transporters and epithelial tight junction proteins in the renal and intestinal tissues were analyzed using an enzyme-linked immunosorbent assay. RESULTS The protein extract of Poecilobdella manillensis lyophilized powder (49 mg/kg) showed an enhanced anti-trioxypurine ability than that of allopurinol (5 mg/kg) (P < 0.05). A total of nine bacterial genera were identified to be closely related to the anti-trioxypurine activity of Poecilobdella manillensis powder, which included the genera of Prevotella , Delftia , Dialister , Akkermansia , Lactococcus , Escherichia_Shigella , Enterococcus , and Bacteroides . Furthermore, 22 metabolites in the serum were found to be closely related to the anti-trioxypurine activity of Poecilobdella manillensis powder, which correlated to the Kyoto Encyclopedia of Genes and Genomes pathways of cysteine and methionine metabolism, sphingolipid metabolism, galactose metabolism, and phenylalanine, tyrosine, and tryptophan biosynthesis. Correlation analysis found that changes in the gut microbiota were significantly related to these metabolites. CONCLUSION The proteins in Poecilobdella manillensis powder were effective for HUA. Mechanistically, they are associated with improvements in gut microbiota dysbiosis and the regulation of sphingolipid and galactose metabolism.

Comparative Metabolomics and Network Pharmacology Analysis Reveal Shared Neuroprotective Mechanisms of Bacopa monnieri (L.) Wettst and Centella asiatica (L.) Urb.

Bacopa monnieri (L.) Wettst and Centella asiatica (L.) Urb., two nootropics, are recognized in Indian Ayurvedic texts. Studies have attempted to understand their action as memory enhancers and neuroprotectants, but many molecular aspects remain unknown. We propose that Bacopa monnieri (L.) Wettst and Centella asiatica (L.) Urb. share common neuroprotective mechanisms. Mass spectrometry-based untargeted metabolomics and network pharmacology approach were used to identify potential protein targets for the metabolites from each extract. Phytochemical analyses and cell culture validation studies were also used to assess apoptosis and ROS activity using aqueous extracts prepared from both herbal powders. Further, docking studies were also performed using the LibDock protocol. Untargeted metabolomics and network pharmacology approach unveiled 2751 shared metabolites and 3439 and 2928 non-redundant metabolites from Bacopa monnieri and Centella asiatica extracts, respectively, suggesting a potential common neuroprotective mechanism among these extracts. Protein-target prediction highlighted 92.4% similarity among the proteins interacting with metabolites for these extracts. Among them, kinases mapped to MAPK, mTOR, and PI3K-AKT signaling pathways represented a predominant population. Our results highlight a significant similarity in the metabolome of Bacopa monnieri (L.) Wettst and Centella asiatica (L.) Urb., and their potential protein targets may be attributed to their common neuroprotective functions.

The Impact of Antenatal Corticosteroids on the Metabolome of Preterm Newborns: An Untargeted Approach.

We analyzed and compared variations in the urinary metabolome, as well as postnatal clinical outcomes among preterm infants, based on the timing of antenatal corticosteroid (ACS) administration in response to preterm labor onset in their mothers. This was a prospective observational study held in the Neonatal Intensive Care Unit, Department of Woman's and Child's Health, Padova University Hospital (Italy). A urine sample was obtained from each patient within 24 h of birth; Mass Spectrometry-based untargeted metabolomics analysis was then conducted. We searched for any significant disparities in the metabolomic profile of preterm newborns subjected to antenatal corticosteroid (ACS) treatment at varying timings; their correlation with clinical outcomes were also evaluated. The group receiving ACS within the optimal time window (1-7 days before delivery) exhibited elevated levels of cysteine, N-acetylglutamine, propionyl carnitine and 5-hydroxyindolacetic acid, coupled with a decrease in pipecolic acid. Clinically, this group demonstrated a reduced need for invasive ventilation (p = 0.04). In conclusion, metabolomics analysis identified several metabolites that discriminated preterm infants whose mothers received ACS within the recommended time window. Elevated levels of cysteine and 5-Hydroxyindoleacetic acid, metabolites characterized by antioxidant and anti-inflammatory properties, were observed in these infants. This metabolic profile correlated with improved respiratory outcomes, as evidenced by a reduced necessity for invasive ventilation at birth.

Exploring potential predictors of Henoch-Schönlein purpura nephritis: a pilot investigation on urinary metabolites.

Henoch-Schönlein purpura nephritis (HSPN) is the most severe manifestation of Henoch-Schönlein purpura (HSP). This study aimed to determine the role of urine metabolomics in predicting HSPN and explore the potential mechanisms of HSP. A liquid chromatography-tandem mass spectrometry-based untargeted metabolomics analysis was performed to investigate the urinary metabolic profiles of 90 participants, comprising 30 healthy children (group CON) and 60 patients with HSP, including 30 HSP patients without renal involvement (group H) and 30 HSPN patients (group HSPN). The differentially expressed metabolites (DEMs) were identified using orthogonal partial least squares discriminant analysis (OPLS-DA), and subsequent bioinformatics analysis was conducted to elucidate the perturbed metabolic pathways. A total of 43 DEMs between H and HSPN groups were analyzed by the Kyoto Encyclopedia of Gene and Genome (KEGG) database, and the result indicates that glycine, serine and threonine metabolism, and cysteine and methionine metabolism were significantly disturbed. A composite model incorporating propionylcarnitine and indophenol sulfate was developed to assess the risk of renal involvement in pediatric patients with HSP. Conclusion: This study reveals the metabolic alterations in healthy children, HSPN patients, and HSP patients without renal involvement. Furthermore, propionylcarnitine and indophenol sulfate may be potential predictive biomarkers of the occurrence of HSPN. What is Known: • HSP is the predominant type of vasculitis observed in children. The long-term prognosis of HSP is contingent upon the extent of renal impairment. In severe nephritis, a delay in appropriate treatment may lead to fibrosis progression and subsequent development of chronic kidney disease (CKD), even leading to renal failure. • The application of metabolomics in investigating diverse renal disorders has been documented. Urine is a robust and sensitive medium for metabolomics detection. What is New: • The metabolic profiles were identified in urine samples of healthy children and those with HSP at the early stage of the disease. Different metabolites were identified between HSP patients without nephritis and those who developed HSPN. • These different metabolites may affect oxidative stress in the progression of HSPN.

Allele-specific dysregulation of lipid and energy metabolism in early-stage hypertrophic cardiomyopathy

IntroductionHypertrophic cardiomyopathy (HCM) results from pathogenic variants in sarcomeric protein genes that increase myocyte energy demand and lead to cardiac hypertrophy. However, it is unknown whether a common metabolic trait underlies cardiac phenotype at the early disease stage. To address this question and define cardiac biochemical pathology in early-stage HCM, we studied two HCM mouse models that express pathogenic variants in cardiac troponin T (Tnt2) or myosin heavy chain (Myh6) genes, and have marked differences in cardiac imaging phenotype, mitochondrial function at early disease stage. MethodsWe used a combination of echocardiography, transcriptomics, mass spectrometry-based untargeted metabolomics (GC-TOF, HILIC, CSH-QTOF), and computational modeling (CardioNet) to examine cardiac structural and metabolic remodeling at early disease stage (5 weeks of age) in R92W-TnT+/− and R403Q-MyHC+/− mutant mice. Data from mutants was compared with respective littermate controls (WT). ResultsAllele-specific differences in cardiac phenotype, gene expression and metabolites were observed at early disease stage. LV diastolic dysfunction was prominent in TnT mutants. Differentially-expressed genes in TnT mutant hearts were predominantly enriched in the Krebs cycle, respiratory electron transport, and branched-chain amino acid metabolism, whereas MyHC mutants were enriched in mitochondrial biogenesis, calcium homeostasis, and liver-X-receptor signaling. Both mutant hearts demonstrated significant alterations in levels of purine nucleosides, trisaccharides, dicarboxylic acids, acylcarnitines, phosphatidylethanolamines, phosphatidylinositols, ceramides and triglycerides; 40.4 % of lipids and 24.7 % of metabolites were significantly different in TnT mutants, whereas 10.4 % of lipids and 5.8 % of metabolites were significantly different in MyHC mutants. Both mutant hearts had a lower abundance of unsaturated long-chain acyl-carnitines (18:1, 18:2, 20:1), but only TnT mutants showed enrichment of FA18:0 in ceramide and cardiolipin species. CardioNet predicted impaired energy substrate metabolism and greater phospholipid remodeling in TnT mutants than in MyHC mutants. ConclusionsOur systems biology approach revealed marked differences in metabolic remodeling in R92W-TnT and R403Q-MyHC mutant hearts, with TnT mutants showing greater derangements than MyHC mutants, at early disease stage. Changes in cardiolipin composition in TnT mutants could contribute to impairment of energy metabolism and diastolic dysfunction observed in this study, and predispose to energetic stress, ventricular arrhythmias under high workloads such as exercise.

Nutrition and flavor analysis of Spirulina through co-fermentation with Lactobacillus acidophilus and Kluyveromyces marxianus and its effect on attenuating metabolic associated fatty liver disease

Spirulina is widely known as the “Super Nutritious Food of the Future” due to its exceptional nutritional properties. In this article, we investigated the effects of fermentation using a combination of Lactobacillus acidophilus KDB-03 and Kluyveromyces marxianus GDMCC 2.119 on Spirulina. Fermentation significantly increased the total flavonoid and phenol levels in Spirulina, and enhanced the 2,2-diphenyl-1-picrylhydrazyl free radical scavenging capacity and ferric-reducing antioxidant power. Untargeted ultra-performance liquid chromatography-electrospray tandem mass spectrometry-based metabolomics and gas chromatography-ion mobility spectrometry analysis revealed alterations in the metabolites and flavor components of fermented Spirulina (FS). Furthermore, a mice experiment demonstrated that FS could effectively alleviate metabolism-associated fatty liver disease (MAFLD) by regulating liver metabolism, intestinal flora, and facilitating communication between the liver and gut. These findings suggest that fermentation could be a innovative approach to enhance the diversity of Spirulina products, and potentially be used as a strategy for the prevention and treatment of MAFLD.

Toxicity of Ethyl Formate to Tribolium castaneum (Herbst) Exhibiting Different Levels of Phosphine Resistance and Its Influence on Metabolite Profiles

Ethyl formate (EF), a naturally occurring fumigant, has attracted widespread attention owing to its low toxicity in mammals. Here, Direct Immersion Solid-Phase Microextraction (DI-SPME) was employed for sample preparation in mass spectrometry-based untargeted metabolomics to evaluate the effects on Tribolium castaneum (Herbst) strains with different levels of PH3 resistance (sensitive, TC-S; moderately resistant, TC-M; strongly resistant, TC-SR) when exposed to a sub-lethal concentration (LC30) of EF. The bioassay indicated that T. castaneum strains with varying PH3 resistance levels did not confer cross-resistance to EF. A metabolomic analysis revealed that exposure to sublethal doses of EF significantly altered 23 metabolites in T. castaneum, including 2 that are unique to the species which remained unaffected by external conditions, while 11 compounds showed a strong response. A pathway topology analysis indicated that EF caused changes to several metabolic pathways, mainly involving fatty acids and their related metabolic pathways. This study showed that EF can induce highly similar metabolic responses in insects across varying levels of PH3 resistance, suggesting that the mechanisms driving the toxicity of EF and PH3 are distinct. These insights significantly extend our knowledge of the toxic mechanisms of EF and provide direct evidence for the efficacy of EF treatment for managing PH3 resistance in insects.

Rolling forms the diversities of small molecular nonvolatile metabolite profile and consequently shapes the bacterial community structure for Keemun black tea

The detailed dynamics of small molecular nonvolatile chemical and bacterial diversities, as well as their relationship are still unclear in the manufacturing process of Keemun black tea (KMBT). Herein, mass spectrometry-based untargeted metabolomics, Feature-based Molecular Networking (FBMN) and bacterial DNA amplicon sequencing were used to investigate the dense temporal samples of the manufacturing process. For the first time, we reveal that the pyrogallol-type catechins are oxidized asynchronously before catechol-type catechins during the black tea processing. Rolling is the key procedure for forming the small molecular nonvolatile metabolite profile (SMNMetProf), increasing the metabolite richness, and then shaping the bacterial community structure in the KMBT manufacturing process, which decreases both molecular weight and molecular polarity of the small molecular nonvolatile metabolites. The SMNMetProf of black tea is formed by the endogenous enzymatic oxidation of tea leaves, rather than bacterial fermentation.

Reduction in creatine metabolites in macrophages exposed to small molecule analogues of the anti-inflammatory parasitic worm product ES-62.

ES-62, a protein secreted by Acanthocheilonema viteae, is anti-inflammatory by virtue of covalently attached phosphorylcholine (PC) residues and thus a library of drug-like small molecule analogues (SMAs) based on its PC moieties has been designed for therapeutic purposes. Two members, SMAs 11a and 12b, were previously found to suppress production of pro-inflammatory cytokines by mouse bone marrow-derived macrophages (BMMs) exposed to cytosine-phosphate-guanosine oligodeoxynucleotides (CpG), agonists for Toll-like receptor 9. In order to explore the mechanism of action underlying such activities, an untargeted mass spectrometry-based metabolomics screen was undertaken. Stimulation of BMMs with CpG produced significant metabolic changes relating to glycolysis and the TCA cycle but the SMAs had little impact on this. Also, the SMAs did not promote alterations in metabolites known to be associated with macrophage M1/M2 polarization. Rather, BMMs exposed to SMAs 11a or 12b prior to CpG treatment, or even alone, revealed downregulation of metabolites of creatine, a molecule whose major role is in the transport of high energy phosphate from the mitochondria to the cytosol. These data therefore provide insight into a possible mechanism of action of molecules with significant therapeutic potential that has not previously been described for parasitic worm products.

The exometabolome of microbial communities inhabiting bare ice surfaces on the southern Greenland Ice Sheet.

Microbial blooms colonize the Greenland Ice Sheet bare ice surface during the ablation season and significantly reduce its albedo. On the ice surface, microbes are exposed to high levels of irradiance, freeze-thaw cycles, and low nutrient concentrations. It is well known that microorganisms secrete metabolites to maintain homeostasis, communicate with other microorganisms, and defend themselves. Yet, the exometabolome of supraglacial microbial blooms, dominated by the pigmented glacier ice algae Ancylonema alaskanum and Ancylonema nordenskiöldii, remains thus far unstudied. Here, we use a high-resolution mass spectrometry-based untargeted metabolomics workflow to identify metabolites in the exometabolome of microbial blooms on the surface of the southern tip of the Greenland Ice Sheet. Samples were collected every 6 h across two diurnal cycles at 5 replicate sampling sites with high similarity in community composition, in terms of orders and phyla present. Time of sampling explained 46% (permutational multivariate analysis of variance [PERMANOVA], pseudo-F = 3.7771, p = 0.001) and 27% (PERMANOVA, pseudo-F = 1.8705, p = 0.001) of variance in the exometabolome across the two diurnal cycles. Annotated metabolites included riboflavin, lumichrome, tryptophan, and azelaic acid, all of which have demonstrated roles in microbe-microbe interactions in other ecosystems and should be tested for potential roles in the development of microbial blooms on bare ice surfaces.

Polygoni Cuspidati Rhizoma et Radix extract attenuates high-fat diet-induced NAFLD in rats: Impact on untargeted serum metabolomics and liver lipidomics

BackgroundPolygoni Cuspidati Rhizoma et Radix (PCRR; the root and rhizome of Polygonum cuspidatum Sieb. et Zucc) has long been used to treat atherosclerosis, gastrointestinal disorders, and liver disease. However, the underlying mechanism of PCRR against liver steatosis remains unclear. PurposeThe aim of this study was to determine the potential therapeutic targets and molecular mechanisms of PCRR in improving liver steatosis. MethodsA high-fat diet (HFD)-induced simple steatosis model in rats was used to evaluate the therapeutic effect of polyphenol-rich water extract from PCRR. Its underlying mechanisms were revealed using untargeted mass spectrometry-based serum metabolomics, hepatic lipidomic profiling approaches, qPCR, and Western blotting assays. ResultsOur study showed that PCRR-treated rats displayed significant reduction of serum TC and LDL-C, hepatic TG, TC levels, and total liver lipid content under a HFD feeding condition. Untargeted metabolomics showed that PCRR significantly restored the overall levels of nine lipid classes in liver tissues and elevated the bile acid levels in the serum samples. We demonstrated that PCRR prevented HFD-induced hepatic steatosis by regulating the expressions of TG metabolism proteins, including p-AMPK, p-ACC, PPARα, and FAS, as well as the expressions of TC metabolism proteins, including p-AMPK, LDLR, and CYP7A1. Moreover, resveratrol and/or polydatin­-derived metabolites were solely detected in the serum samples of the PCRR-treated group, indicating these microbial transformed metabolites might partly contribute to the beneficial effects of PCRR. ConclusionPCRR can be employed as a therapeutic agent to improve non-alcoholic fatty liver disease via regulating lipid metabolism.

Discovering Nature's shield: Metabolomic insights into green zinc oxide nanoparticles Safeguarding Brassica parachinensis L. from cadmium stress

Heavy metal cadmium (Cd) hinders plants' growth and productivity by causing different morphological and physiological changes. Nanoparticles (NPs) are promising for raising plant yield and reducing Cd toxicity. Nonetheless, the fundamental mechanism of nanoparticle-interfered Cd toxicity in Brassica parachineses L. remains unknown. A novel ZnO nanoparticle (ZnO-NPs) was synthesized using a microalgae strain (Chlorella pyrenoidosa) through a green process and characterized by different standard parameters through TEM, EDX, and XRD. This study examines the effect of different concentrations of ZnO-NPs (50 and 100 mgL−1) in B. parachineses L. under Cd stress through ultra-high-performance liquid chromatography/high-resolution mass spectrometry-based untargeted metabolomics profiling. In the presence of Cd toxicity, foliar spraying with ZnO-NPs raised Cu, Fe, Zn, and Mg levels in the roots and/or leaves, improved seedling development, as demonstrated by increased plant height, root length, and shoot and root fresh weight. Furthermore, the ZnO-NPs significantly enhanced the photosynthetic pigments and changed the antioxidant activities of the Cd-treated plants. Based on a metabolomics analysis, 481 untargeted metabolites were accumulated in leaves under normal and Cd-stressed conditions. These metabolites were highly enriched in producing organic acids, amino acids, glycosides, flavonoids, nucleic acids, and vitamin biosynthesis. Surprisingly, ZnO-NPs restored approximately 60% of Cd stress metabolites to normal leaf levels. Our findings suggest that green synthesized ZnO-NPs can balance ions’ absorption, modulate the antioxidant activities, and restore more metabolites associated with plant growth to their normal levels under Cd stress. It can be applied as a plant growth regulator to alleviate heavy metal toxicity and improve crop yield in heavy metal-contaminated regions.

Mass spectrometry-based untargeted metabolomics approaches for comprehensive structural annotation of bioactive metabolites from bushy cashew (Anacardium humile) fruits

Anacardium humile (bushy cashew) is a native Brazilian plant with substantial pharmacological potential and noteworthy commercial significance in the food industry. This study introduces an untargeted metabolomics approach based on mass spectrometry for the comprehensive structural annotation of bioactive metabolites. The fruits were collected from three distinct sites and subjected to LC-HRMS/MS analysis. A total of eighty-eight compounds were putatively annotated across various metabolite classes. This unveiled a metabolic profile characterized by notable concentrations of polyphenols, including flavonoids, tannins, phenolic acids, and quinones, while aliphatic acids and terpenes were found in limited quantities. Noteworthy, no significant disparities in the metabolic content were observed among the collection sites. The three principal metabolites (peonidin 3-O-glucopyranoside, methylcyanidin and methyldelphinidin) underwent assessment for antioxidant activity via molecular docking analysis, subsequently generating structure-based feature pharmacophores. The process of annotation propagation yielded a comprehensive qualitative appraisal of the A. humile metabolome. The outcomes obtained offer potential candidates for further exploration of their nutraceutical attributes.

Dynamic Changes in the Microbial Community and Metabolite Profile during the Pile Fermentation Process of Fuzhuan Brick Tea.

The pile fermentation process of Fuzhuan brick tea is unique in that it involves preheating without the use of starter cultures. The detailed metabolite changes and their drivers during this procedure are not known. Characterizing these unknown changes that occur in the metabolites and microbes during pile fermentation of Fuzhuan brick tea is important for industrial modernization of this traditional fermented food. Using microbial DNA amplicon sequencing, mass spectrometry-based untargeted metabolomics, and feature-based molecular networking, we herein reveal that significant changes in the microbial community occur before changes in the metabolite profile. These changes were characterized by a decrease in Klebsiella and Aspergillus, alongside an increase in Bacillus and Eurotium. The decrease in lysophosphatidylcholines, unsaturated fatty acids, and some astringent flavan-3-ols and bitter amino acids, as well as the increase in some less astringent flavan-3-ols and sweet or umami amino acids, contributed importantly to the overall changes observed in the metabolite profile. The majority of these changes was caused by bacterial metabolism and the corresponding heat generated by it.

Metabolomic Diversity in Microbial Mats Under Different Environmental Conditions: A Tool to Test Microbial Ecosystem Chemical Change.

Microbial mats are microbial communities capable of recycling the essential elements of life and considered to be the oldest evidence of microbial communities on Earth. Due to their uniqueness and limited sampling material, analyzing their metabolomic profile in different seasons or conditions is challenging. In this study, microbial mats from a small pond in the Cuatro Cienegas Basin in Coahuila, Mexico, were collected in wet and dry seasons. In addition to these samples, mesocosm experiments from the wet samples were set. These mats are elastic and rise after heavy rainfall by forming gas domes structures known as "Archean domes", by the outgassing of methanogenic bacteria, archaea, and sulfur bacteria. Extracts from all mats and mesocosms were subjected to untargeted mass spectrometry-based metabolomics and molecular networking analysis. Interestingly, each mat showed high chemical diversity that may be explained by the temporal dynamic processes in which they were sampled.

Unlocking Potentially Therapeutic Phytochemicals in Capadulla (Doliocarpus dentatus) from Guyana Using Untargeted Mass Spectrometry-Based Metabolomics.

Doliocarpus dentatus is thought to have a wide variety of therapeutic phytochemicals that allegedly improve libido and cure impotence. Although a few biomarkers have been identified with potential antinociceptive and cytotoxic properties, an untargeted mass spectrometry-based metabolomics approach has never been undertaken to identify therapeutic biofingerprints for conditions, such as erectile dysfunction, in men. This study executes a preliminary phytochemical screening of the woody vine of two ecotypes of D. dentatus with renowned differences in therapeutic potential for erectile dysfunction. Liquid chromatography-mass spectrometry-based metabolomics was used to screen for flavonoids, terpenoids, and other chemical classes found to contrast between red and white ecotypes. Among the metabolite chemodiversity found in the ecotype screens, using a combination of GNPS, MS-DIAL, and SIRIUS, approximately 847 compounds were annotated at levels 2 to 4, with the majority of compounds falling under lipid and lipid-like molecules, benzenoids and phenylpropanoids, and polyketides, indicative of the contributions of the flavonoid, shikimic acid, and terpenoid biosynthesis pathways. Despite the extensive annotation, we report on 138 tentative compound identifications of potentially therapeutic compounds, with 55 selected compounds at a level-2 annotation, and 22 statistically significant therapeutic biomarkers, the majority of which were polyphenols. Epicatechin methyl gallate, catechin gallate, and proanthocyanidin A2 had the greatest significant differences and were also relatively abundant among the red and white ecotypes. These putatively identified compounds reportedly act as antioxidants, neutralizing damaging free radicals, and lowering cell oxidative stress, thus aiding in potentially preventing cellular damage and promoting overall well-being, especially for treating erectile dysfunction (ED).

Optimization Strategies for Mass Spectrometry-Based Untargeted Metabolomics Analysis of Small Polar Molecules in Human Plasma.

The untargeted approach to mass spectrometry-based metabolomics has a wide potential to investigate health and disease states, identify new biomarkers for diseases, and elucidate metabolic pathways. All this holds great promise for many applications in biological and chemical research. However, the complexity of instrumental parameters on advanced hybrid mass spectrometers can make the optimization of the analytical method immensely challenging. Here, we report a strategy to optimize the selected settings of a hydrophilic interaction liquid chromatography-tandem mass spectrometry method for untargeted metabolomics studies of human plasma, as a sample matrix. Specifically, we evaluated the effects of the reconstitution solvent in the sample preparation procedure, the injection volume employed, and different mass spectrometry-related operating parameters including mass range, the number of data-dependent fragmentation scans, collision energy mode, duration of dynamic exclusion time, and mass resolution settings on the metabolomics data quality and output. This study highlights key instrumental variables influencing the detection of metabolites along with suggested settings for the IQ-X tribrid system and proposes a new methodological framework to ensure increased metabolome coverage.

Qualitative metabolomics-based characterization of a phenolic UDP-xylosyltransferase with a broad substrate spectrum from Lentinus brumalis

Wood-decaying fungi are the major decomposers of plant litter. Heavy sequencing efforts on genomes of wood-decaying fungi have recently been made due to the interest in their lignocellulolytic enzymes; however, most parts of their proteomes remain uncharted. We hypothesized that wood-decaying fungi would possess promiscuous enzymes for detoxifying antifungal phytochemicals remaining in the dead plant bodies, which can be useful biocatalysts. We designed a computational mass spectrometry-based untargeted metabolomics pipeline for the phenotyping of biotransformation and applied it to 264 fungal cultures supplemented with antifungal plant phenolics. The analysis identified the occurrence of diverse reactivities by the tested fungal species. Among those, we focused on O-xylosylation of multiple phenolics by one of the species tested, Lentinus brumalis. By integrating the metabolic phenotyping results with publicly available genome sequences and transcriptome analysis, a UDP-glycosyltransferase designated UGT66A1 was identified and validated as an enzyme catalyzing O-xylosylation with broad substrate specificity. We anticipate that our analytical workflow will accelerate the further characterization of fungal enzymes as promising biocatalysts.

Mass spectrometry-based untargeted metabolomics study of non-obese individuals with non-alcoholic fatty liver disease

Objectives Non-alcoholic fatty liver disease (NAFLD) is a disease characterized by the accumulation of excessive fat in the liver, which can lead to fibrosis and has an increasing prevalence. NAFLD requires non-invasive diagnostic biomarkers. While typically observed in overweight individuals, it can also occur in non-obese/non-overweight individuals. Comparative studies on non-obese NAFLD patients are scarce. This study aimed to conduct a using liquid chromatography-high resolution mass spectrometry (LC-MS/MS)-based metabolic profiling of non-obese NAFLD patients and healthy controls. Materials and methods The patient group consisted of 27 individuals with NAFLD, while the healthy control group included 39 individuals. Both groups were between 18 and 40 years old, had a BMI of less than 25 and had alcohol consumption less than 20 g/week for men and 10 g/week for women. Serum samples were collected and analyzed using LC-MS/MS. The data were analyzed using the TidyMass and MetaboAnalyst. Results The LC-MS/MS analyses detected significant changes in D-amino acid metabolism, vitamin B6 metabolism, apoptosis, mTOR signaling pathway, lysine degradation, and phenylalanine metabolism pathways in non-obese NAFLD patients. Significant changes were also observed in the metabolites D-pantothenic acid, hypoxanthine, citric acid, citramalic acid, L-phenylalanine, glutamine, and histamine-trifluoromethyl-toluidide, β-hydroxymyristic acid, DL-Lactic acid, and 3-methyl-2-oxopentanoic. Overall, the study provides valuable insights into the metabolic changes associated with non-obese NAFLD patients and can contribute to the development of non-invasive diagnostic biomarkers for NAFLD. Conclusions This study sheds light on the metabolic changes in non-obese NAFLD patients. Further research is needed to better understand the metabolic changes associated with NAFLD and to develop effective treatment options.

Chemical Modification of a Bacterial Siderophore by a Competitor in Dual-Species Biofilms.

Chemical communication between competing bacteria in multi-species environments often enables both species to adapt and survive, and perhaps even thrive. P. aeruginosa and S. aureus are two bacterial pathogens found in natural biofilms, especially in the lungs of cystic fibrosis (CF) patients, where recent studies showed that there is often cooperation between the two species, leading to increased disease severity and antibiotic resistance. However, the mechanisms behind this cooperation are poorly understood. In this study, we analyzed co-cultured biofilms in various settings, and we applied untargeted mass spectrometry-based metabolomics analyses, combined with synthetic validation of candidate compounds. We unexpectedly discovered that S. aureus can convert pyochelin into pyochelin methyl ester, an analogue of pyochelin with reduced affinity for iron (III). This conversion allows S. aureus to coexist more readily with P. aeruginosa and unveils a mechanism underlying the formation of robust dual-species biofilms.