LC-MS Metabolomics Research Articles

Study on the Differences in Fecal Metabolites and Microbial Diversity of Jiangshan Black-Bone Chickens with Different Earlobe Colors

The differences in earlobe color of Jiangshan black-bone chickens have been reported to be caused by the combined effects of melanin and collagen. In this study, we conducted LC-MS untargeted metabolomics and 16S rDNA diversity sequencing on the cecal contents of two types of earlobes: peacock green (Blue and Green group) and dark reddish-purple (Black group). The metabolomic sequencing identified a total of 747 differential metabolites (DMs), in which the metabolites were primarily enriched in tyrosine and tryptophan metabolism pathways between peacock green and dark reddish-purple earlobes. There were 15 different bacterial taxa among the three groups of earlobes at the genus level, and correlation analysis between metabolites and microbes revealed that the DMs between peacock green and dark reddish-purple earlobes were positively correlated with the different bacterial taxa. In short, there are differences in gut microbiota and metabolites between Jiangshan black-bone chickens with peacock green earlobes and those with dark reddish-purple earlobes. Our results suggest that the bacterial phyla Firmicutes and Bacteroidota may influence melanin synthesis by affecting tryptophan metabolism, induced by 5-Methoxyindoleacetate, and tyrosine metabolism, induced by maleylacetoacetic acid and maleic acid, leading to differences in earlobe color.

Relationship between pyrimidines, purines and fatty acids in milk of dairy cows fed distinct carbohydrate types: a metabolomic approach

The aim of this research was to evaluate changes in the milk metabolome of dairy cows fed different carbohydrate types from silages and concentrates, with special focus on purines and pyrimidines, likely associated with the microbial activity in the rumen. Furthermore, the relationship between the milk fatty acids and pyrimidine and purine metabolism retrieved in milk was investigated for potential correlations between these 2 groups of potential milk biomarkers. Twenty-four lactating dairy cows were used in a crossover design, where each cow received 2 of 4 diets. Diets were formulated based on a 2 by 2 factorial arrangement, using either grass-clover or corn silage as a forage source, and barley or dried beet pulp in the concentrate fraction. Fatty acids and LC-MS metabolome of the milk were analyzed. The milk metabolome and fatty acid concentration could differentiate the 4 diets used in the trial, while the milk metabolome alone could only differentiate the 2 silage types. Milk odd-chain fatty acids (C17:1 and C17:0) were correlated with pyrimidine and purine derivatives, with uric acid being significantly higher in milk samples associated with corn silage diets. Therefore, the results indicate potential differences in microbial protein synthesis between the diets, but a validation with actual ruminal microbial protein synthesis and investigation in digestive biofluids are necessary for further confirmation.

Impact of Lentinus sajor-caju on Lignocellulosic Biomass, In Vitro Rumen Digestibility and Antioxidant Properties of Astragalus membranaceus var. mongholicus Stems under Solid-State Fermentation Conditions

Lentinus sajor-caju has shown potential for the bioconversion of lignocellulosic biomass in various substrates. Here, we evaluated whether L. sajor-caju affects the lignocellulosic biomass, in vitro fermentation and antioxidant properties of Astragalus membranaceus var. mongholicus (AMM) stems. The lignocellulosic biomass content and the antioxidant activity of AMM stems were determined by scanning electron microscopy, chemical component analysis, in vitro fermentation and LC-MS metabolomics after 30 days of fermentation by L. sajor-caju at 25 °C. L. sajor-caju significantly altered the rigid structure of the stems. Compared with the control condition, the lignocellulose contents were significantly reduced (p < 0.001) and improved in vitro digestibility and total volatile fatty acids. In total, 624 differential metabolites, including 201 up-regulated and 423 down-regulated, were identified in unfermented and fermented comparison groups. Correlation analysis indicated that there were strongly correlations of the total phenolic content and total antioxidant capacity. Meanwhile, the differential metabolites were primarily associated with antioxidant activity, with 4(3H)-quinazolinone, dihydrocarvyl acetate and jaceoside being the most representative. In summary, L. sajor-caju altered the composition of the cell wall of AMM stems, thereby enhancing their antioxidant activity.

Investigating the modulatory effects of lactoferrin on depressed rats through 16S rDNA gene sequencing and LC-MS metabolomics analysis.

Lactoferrin is a natural multifunctional glycoprotein with potential antidepressant-like effects. However, the mechanism of its antidepressant effect has not been explored from the perspective of gut flora metabolism. Therefore, we employed both 16S rDNA gene sequencing and LC-MS metabolomics analysis to investigate the regulatory effects and mechanisms of lactoferrin in a rat model of depression. After one week of acclimatization, twenty-four 7-week-old male Sprague-Dawley rats were randomly and equally assigned into three groups: the control group, the model group, and the lactoferrin intervention group. The control group rats were housed under standard conditions, while the rats in the model and lactoferrin intervention groups were individually housed and exposed to chronic unpredictable mild stress for 44days simultaneously. The lactoferrin intervention group was provided with water containing 2% lactoferrin (2g/100ml). Behavioural tests were conducted at week 7. Upon completion of the behavioral tests, the rats were anesthetized with isoflurane, humanely euthanized using a rat guillotine, and tissue samples were collected for further experiments. The results indicated that lactoferrin intervention led to an increase in sucrose solution consumption, horizontal movement distance, number of cross platforms, and residence time in the target quadrant. Additionally, it resulted in an increase in jejunal tight junction protein ZO-1 expression and a suppression of serum expression of inflammatory factors, Lipopolysaccharide and Diamine oxidase. In summary, lactoferrin can regulate the metabolic disorder of intestinal flora, reduce intestinal permeability, and further regulate the metabolic balance of hippocampal tissues through the microbiota-gut-brain axis. This process ultimately alleviates the depression-like behavior in rats.

Urinary Biomarkers of Strawberry and Blueberry Intake.

Introduction There is increasing interest in food biomarkers to address the shortcomings of self-reported dietary assessments. Berries are regarded as important fruits worldwide; however, there are no well-validated biomarkers of berry intake. Thus, the objective of this study is to identify urinary biomarkers of berry intake. Methods For the discovery study, participants consumed 192 g strawberries with 150 g blueberries, and urine samples were collected at 2, 4, 6, and 24 h post-consumption. A dose-response study was performed, whereby participants consumed three portions (78 g, 278 g, and 428 g) of mixed strawberries and blueberries. The urine samples were profiled by an untargeted LC-MS metabolomics approach in the positive and negative modes. Results Statistical analysis of the data revealed that 39 features in the negative mode and 15 in the positive mode significantly increased between fasting and 4 h following mixed berry intake. Following the analysis of the dose-response data, 21 biomarkers showed overall significance across the portions of berry intake. Identification of the biomarkers was performed using fragmentation matches in the METLIN, HMDB, and MoNA databases and in published papers, confirmed where possible with authentic standards. Conclusions The ability of the panel of biomarkers to assess intake was examined, and the predictability was good, laying the foundations for the development of biomarker panels.

LC-MS untargeted metabolomics reveals metabolic disturbance and ferroptosis in MWCNTs-induced hepatotoxicity of Cyprinus carpio

In recent years, there is a great concern about the potential adverse effects of carbon nanotubes (CNTs) on the aquatic systems due to their increasingly extensive application. In this study, juvenile Cyprinus carpio were exposed to multi-walled CNTs (MWCNTs) at concentrations of 0, 0.25, and 2.5 mg L-1 for 28 days. Then, oxidative stress indicators and metabolite profile of the livers were assessed. Results showed the significant increase of malondialdehyde (MDA) content and decrease of glutathione (GSH) activities in fish treated with 2.5 mg L-1 MWCNTs. LC-MS untargeted metabolomics demonstrated that 406 and 274 metabolites in fish treated with 2.5 mg L-1 MWCNTs were significantly up- and down-regulated, respectively. KEGG functional annotation analysis showed the disturbance of amino acid metabolism, lipid metabolism, and nucleotide metabolism. In addition, ferroptosis signaling pathway was detected. Therefore, iron content analysis and quantitative real-time RT-PCR assay were performed furtherly to validate the contribution of ferroptosis to MWCNTs-induced hepatotoxicity. The iron content increased significantly and the mRNA levels of ferroptosis-related genes including STEAP3, ACSL4, NCOA4, TFR1, NRF2, SLC3A2, SLC7A11, GPX4, and FPN1 were also obviously changed. Taken together, our study suggested that MWCNTs exposure-induced ferroptosis were associated with iron overload and lipid peroxidation via NRF2/SLC7A11/GSH/GPX4 axis. Our findings provide essential information to understand the mechanism of CNTs-induced hepatotoxicity in fish and explore potential biomarkers.

Ningxiang Pig-Derived Microbiota Affects the Growth Performance, Gut Microbiota, and Serum Metabolome of Nursery Pigs.

The gut microbiota is crucial for maintaining the host's intestinal homeostasis and metabolism. This study investigated the effects of fecal microbiota transplantation (FMT) from Ningxiang pigs on the growth performance, fecal microbiota, and serum metabolites of the same-old DLY pigs. The results indicated that the average daily gain of FMT pigs was significantly greater than that of the control (CON) group. Compared to the CON group, the FMT group significantly improved the apparent digestibility of crude fiber, crude ash, gross energy, and calcium of the pigs. The analysis of serum antioxidant status revealed that the activities of total superoxide dismutase and catalase in the serum of pigs in the FMT group were significantly elevated, whereas the level of malondialdehyde was significantly reduced. Furthermore, 16S rRNA sequencing analysis revealed that the Ningxiang pig-derived microbiota altered the fecal microbiota structure and modulated the diversity of the gut microbiota in the DLY pigs. Untargeted LC-MS metabolomics demonstrated that pigs in the FMT group exhibited distinct metabolomic profiles compared to those in the CON group. Significant changes were observed in key metabolites involved in amino acid, lipid, and carbohydrate metabolism. Additionally, a correlation analysis between serum differential metabolites and the gut microbiota revealed that the relative abundance of Lachnospiraceae_NK4A136_group and Corynebacterium was highly correlated with lipid compounds. In conclusion, Ningxiang pig-derived microbiota can alleviate oxidative stress and enhance growth performance in DLY pigs by modulating their gut microbiota and metabolic features.

The salivary metabolome of children and parental caregivers in a large-scale family environment study

Human metabolism is complex, and is impacted by genetics, cohabitation, diet, health, and environmental inputs. As such, we applied untargeted LC-MS metabolomics to 1425 saliva samples from a diverse group of elementary school-aged children and their caregivers collected during the Family Life Project, of which 1344 were paired into caregiver/child dyads. We compared metabolomes within and between homes, performed population-wide “metabotype” analyses, and measured associations between metabolites and salivary biomeasures of inflammation, antioxidant potential, environmental tobacco smoke (ETS) exposure, metabolic regulation, and heavy metals. Children and caregivers had similar salivary metabolomes, and dyad explained most metabolomic variation. Our data clustered into two groups, indicating that “metabotypes” exist across large populations. Lastly, several metabolites—putative oxidative damage-associated or pathological markers—were correlated with the above-mentioned salivary biomeasures and heavy metals. Implications of the family environment’s effects on metabolomic variation at population, dyadic, and individual levels for human health are discussed.

Wheat domestication alters root metabolic functions to drive the assembly of endophytic bacteria.

The domestication process progressively differentiated wild relatives from modern cultivars, thus impacting plant-associated microorganisms. Endophytic bacterial communities play vital roles in plant growth, development, and health, which contribute to the crop's sustainable development. However, how plant domestication impacts endophytic bacterial communities and relevant root exudates in wheat remains unclear. First, we have observed that the domestication process increased the root endophytic microbial community diversity of wheat while decreasing functional diversity. Second, domestication decreased the endophytic bacterial co-occurrence network stability, and it did significantly alter the abundances of core microorganisms or potential probiotics. Third, untargeted LC-MS metabolomics revealed that domestication significantly altered the metabolite profiles, and the abundances of various root exudates released were significantly correlated with keystone taxa including the Chryseobacterium, Massilia, and Lechevalieria. Moreover, we found that root exudates, especially L-tyrosine promote the growth of plant-beneficial bacteria, such as Chryseobacterium. Additionally, with L-tyrosine and Chryseobacterium colonized in the roots, the growth of wild wheat's roots was significantly promoted, while no notable effect could be found in the domesticated cultivars. Overall, this study suggested that wild wheat as a key germplasm material, and its native endophytic microbes may serve as a resource for engineering crop microbiomes to improve the morphological and physiological traits of crops in widely distributed poor soils.

Characterising the urinary acylcarnitine and amino acid profiles of HIV/TB co-infection, using LC–MS metabolomics

IntroductionThe human immunodeficiency virus (HIV) and tuberculosis (TB) co-infection presents significant challenges due to the complex interplay between these diseases, leading to exacerbated metabolic disturbances. Understanding these metabolic profiles is crucial for improving diagnostic and therapeutic approaches.ObjectiveThis study aimed to characterise the urinary acylcarnitine and amino acid profiles, including 5-hydroxyindoleacetic acid (5-HIAA), in patients co-infected with HIV and TB using targeted liquid chromatography mass spectrometry (LC–MS) metabolomics.MethodsUrine samples, categorised into HIV, TB, HIV/TB co-infected, and healthy controls, were analysed using HPLC–MS/MS. Statistical analyses included one-way ANOVA and a Kruskal-Wallis test to determine significant differences in the acylcarnitine and amino acid profiles between groups.ResultsThe study revealed significant metabolic alterations, especially in TB and co-infected groups. Elevated levels of medium-chain acylcarnitines indicated increased fatty acid oxidation, commonly associated with cachexia in TB. Altered amino acid profiles suggested disruptions in protein and glucose metabolism, indicating a shift towards diabetes-like metabolic states. Notably, TB was identified as a primary driver of these changes, affecting protein turnover, and impacting energy metabolism in co-infected patients.ConclusionThe metabolic profiling of HIV/TB co-infection highlights the profound impact of TB on metabolic pathways, which may exacerbate the clinical complexities of co-infection. Understanding these metabolic disruptions can guide the development of targeted treatments and improve management strategies, ultimately enhancing the clinical outcomes for these patients. Further research is required to validate these findings and explore their implications in larger, diverse populations.

Comprehensive metabolome characterization of leaves, internodes, and aerial roots of Vanilla planifolia by untargeted LC-MS and GC × GC-MS.

Untargeted metabolomics is a powerful tool that provides strategies for gaining a systematic understanding of quantitative changes in the levels of metabolites, especially when combining different metabolomic platforms. Vanilla is one of the world's most popular flavors originating from cured pods of the orchid Vanilla planifolia. However, only a few studies have investigated the metabolome of V. planifolia, and no LC-MS or GC-MS metabolomics studies with respect to leaves have been performed. The aim of the study was to comprehensively characterize the metabolome of different organs (leaves, internodes, and aerial roots) of V. planifolia. Characterization of the metabolome was achieved using two complementary platforms (GC × GC-MS, LC-QToF-MS), and metabolite identification was based on a comparison with in-house databases or curated external spectral libraries. In total, 127 metabolites could be identified with high certainty (confidence level 1 or 2) including sugars, amino acids, fatty acids, organic acids, and amines/amides but also secondary metabolites such as vanillin-related metabolites, flavonoids, and terpenoids. Ninty-eight metabolites showed significantly different intensities between the plant organs. Most strikingly, aglycons of flavonoids and vanillin-related metabolites were elevated in aerial roots, whereas its O-glycoside forms tended to be higher in leaves and/or internodes. This suggests that the more bioactive aglycones may accumulate where preferably needed, e.g. for defense against pathogens. The results derived from the study substantially expand the knowledge regarding the vanilla metabolome forming a valuable basis for more targeted investigations in future studies, e.g. towards an optimization of vanilla plant cultivation.

Effects of different post-harvest processing methods on changes in the active ingredients of licorice based on LC-MS and plant metabolomics.

Licorice, the dried roots and rhizomes of the Glycyrrhiza uralensis Fisch., holds a prominent status in various formulations within the realm of Chinese medicinal practices. The traditional processing methods of licorice hinder quality assurance, thus prompting Chinese medicine researchers to focus on the fresh processing methods to enhancing processing efficiency and quality. This study aimed to identify the differential compounds of licorice between traditional and fresh processing methods and provide a scientific basis for the fresh processing of licorice and for further research on the processing mechanism. A methodology integrating ultra-performance liquid chromatography with quadrupole-time-of-flight tandem mass spectrometry combined with multivariate statistical analysis was employed to characterize the differential compounds present in licorice between traditional processing and fresh processing. The results derived from principal component analysis and heat map analyses underscored significant differences in the content of bioactive compounds between the two processing methods. By applying conditions of VIP > 1.5 and p < 0.05, a total of 38 differential compounds were identified through t tests, and the transformation mechanisms of select compounds were illustrated. The adoption of fresh processing techniques not only improved processing efficiency but also significantly enhanced the preservation of bioactive compounds within licorice. This research has established a rapid and efficient analytical method for the identification of differential compounds present in differently processed licorice products.

Novel Processing Technology Influences on Nutrient and Flavor Compounds in Fruit Juices, as Assessed via LC-MS Metabolomics

Novel Processing Technology Influences on Nutrient and Flavor Compounds in Fruit Juices, as Assessed via LC-MS Metabolomics

PANAMA-enabled high-sensitivity dual nanoflow LC-MS metabolomics and proteomics analysis

High-sensitivity nanoflow liquid chromatography (nLC) is seldom employed in untargeted metabolomics because current sample preparation techniques are inefficient at preventing nanocapillary column performance degradation. Here, we describe an nLC-based tandem mass spectrometry workflow that enables seamless joint analysis and integration of metabolomics (including lipidomics) and proteomics from the same samples without instrument duplication. This workflow is based on a robust solid-phase micro-extraction step for routine sample cleanup and bioactive molecule enrichment. Our method, termed proteomic and nanoflow metabolomic analysis (PANAMA), improves compound resolution and detection sensitivity without compromising the depth of coverage as compared with existing widely used analytical procedures. Notably, PANAMA can be applied to a broad array of specimens, including biofluids, cell lines, and tissue samples. It generates high-quality, information-rich metabolite-protein datasets while bypassing the need for specialized instrumentation.

MetaboLink: A web application for Streamlined Processing and Analysis of Large-Scale Untargeted Metabolomics Data.

The post-processing and analysis of large-scale untargeted metabolomics data face significant challenges due to the intricate nature of correction, filtration, imputation, and normalization steps. Manual execution across various applications often leads to inefficiencies, human-induced errors, and inconsistencies within the workflow. Addressing these issues, we introduce MetaboLink, a novel web application designed to process LC-MS metabolomics datasets combining established methodologies and offering flexibility and ease of implementation. It offers visualization options for data interpretation, an interface for statistical testing, and integration with PolySTest for further tests and with VSClust for clustering analysis. Fully functional tool is publicly available at https://computproteomics.bmb.sdu.dk/Metabolomics/. The source code is available at https://github.com/anitamnd/MetaboLink and a detailed description of the app can be found at https://github.com/anitamnd/MetaboLink/wiki. A tutorial video can be found at https://youtu.be/ZM6j10S6Z8Q. Supplementary data are available at Bioinformatics online.

Leptochelins A-C, Cytotoxic Metallophores Produced by Geographically Dispersed Leptothoe Strains of Marine Cyanobacteria.

Metals are important cofactors in the metabolic processes of cyanobacteria, including photosynthesis, cellular respiration, DNA replication, and the biosynthesis of primary and secondary metabolites. In adaptation to the marine environment, cyanobacteria use metallophores to acquire trace metals when necessary as well as to reduce potential toxicity from excessive metal concentrations. Leptochelins A-C were identified as structurally novel metallophores from three geographically dispersed cyanobacteria of the genus Leptothoe. Determination of the complex structures of these metabolites presented numerous challenges, but they were ultimately solved using integrated data from NMR, mass spectrometry and deductions from the biosynthetic gene cluster. The leptochelins are comprised of halogenated linear NRPS-PKS hybrid products with multiple heterocycles that have potential for hexadentate and tetradentate coordination with metal ions. The genomes of the three leptochelin producers were sequenced, and retrobiosynthetic analysis revealed one candidate biosynthetic gene cluster (BGC) consistent with the structure of leptochelin. The putative BGC is highly homologous in all three Leptothoe strains, and all possess genetic signatures associated with metallophores. Postcolumn infusion of metals using an LC-MS metabolomics workflow performed with leptochelins A and B revealed promiscuous binding of iron, copper, cobalt, and zinc, with greatest preference for copper. Iron depletion and copper toxicity experiments support the hypothesis that leptochelin metallophores may play key ecological roles in iron acquisition and in copper detoxification. In addition, the leptochelins possess significant cytotoxicity against several cancer cell lines.

Mycobacterium dormancy and antibiotic tolerance within the retinal pigment epithelium of ocular tuberculosis.

Understanding the metabolic environment within the retinal pigment epithelium (RPE) cells altered by infection with Mycobacterium tuberculosis and mycobacterial dormancy is crucial to identify new therapeutic methods to cure ocular tuberculosis. The present study showed that RPE cellular metabolism is altered to foster intracellular M. tuberculosis to enter into the dormant and drug-tolerant state, thereby blunting the efficacy of anti-tuberculosis chemotherapy. RPE cells serve as an anatomical niche as the cells protect invading bacilli from antibiotic treatment. LC-MS metabolomics of RPE cells after co-treatment with H2O2 and M. tuberculosis infection showed that the intracellular environment within RPE cells is enriched with a greater level of oxidative stress. The antibiotic tolerance of intracellular M. tuberculosis within RPE cells can be restored by a metabolic manipulation strategy such as co-treatment of antibiotic with the most downstream glycolysis metabolite, phosphoenolpyruvate.

LC-MS metabolomics and molecular docking approaches to identify antihyperglycemic and antioxidant compounds from Melastoma malabathricum L. Leaf

The dried leaves of Melastoma malabathricum L., locally named Karamunting or Senduduk, is traditionally consumed in many regions in Indonesia as herbal tea to cure different illnesses, including diabetes. To date, information on the compounds responsible for their antidiabetic activity is still very rare. The study aimed to identify bioactive compounds of M. malabathricum L. leaves using LC-MS based metabolomics and molecular docking approaches. The leaves brewed with different methods were subjected to LC−MS measurements and several bioactivity tests (in vivo and in vitro antihyperglycemic, and in vitro antioxidant). LC−MS data were linked to the activity data using multivariate data analysis. Molecular docking using alpha-glucosidase, alpha-amylase, and insulin receptor as protein targets was used to verify the results and study the interaction between the identified compound and protein targets. As results, isoquercetin and myricitrin were identified as compounds strongly associated with alpha-amylase inhibitors, while rutin and epicatechin were identified as alpha-glucosidase inhibitors. Quercitrin, citric acid, quercetin, epicatechin, isoquercitrin, and 7-hydroxycoumarine were strongly correlated with both antihyperglycemic and antioxidant activities. The results of metabolomics were confirmed with molecular docking studies, which showed that some of these compounds acted as competitive inhibitors, while others acted as non-competitive ones. Possible synergism between epicatechin and citric acid in their interaction with IR was detected. Metabolomics combined with molecular docking efficiently identified and confirmed several antihyperglycemic and antioxidant compounds from M. malabathricum L., leaf. This study provides scientific evidence for the traditional use of M. malabathricum L. as an antidiabetic herbal.

Nutrient and metabolite characteristics of the husk, bran and millet isolated from the foxtail millet (Setaria italica L.) during polishing

The utilization of byproducts from foxtail millet polishing can reduce food loss and waste. Thus, it is necessary to know the chemical compounds from the millet and the segregation of the layers. The nutrients including minerals were compared among the husk, bran, and millet, and a LC-MS metabolomics analysis was also performed among them. The results showed that the protein, crude fat and 4 fatty acids, seven minerals, the nitrogen-containing compounds and phenolic acids were at much higher levels in the bran part than the husk and millet, whereas the husk only contained higher levels of dietary fibre, and some minerals. The millet section, as the edible part, contained the lowest level of chemical constituents. It illustrated that the bran part contained more functional and nutritional components than the millet and husk part. Therefore, the bran of the foxtail millet should be a food resources instead of wasting.

Common data models to streamline metabolomics processing and annotation, and implementation in a Python pipeline.

To standardize metabolomics data analysis and facilitate future computational developments, it is essential to have a set of well-defined templates for common data structures. Here we describe a collection of data structures involved in metabolomics data processing and illustrate how they are utilized in a full-featured Python-centric pipeline. We demonstrate the performance of the pipeline, and the details in annotation and quality control using large-scale LC-MS metabolomics and lipidomics data and LC-MS/MS data. Multiple previously published datasets are also reanalyzed to showcase its utility in biological data analysis. This pipeline allows users to streamline data processing, quality control, annotation, and standardization in an efficient and transparent manner. This work fills a major gap in the Python ecosystem for computational metabolomics.