GNPS - Stable-isotope-assisted metabolomics: Deoxynivalenol-stress-related Tyrosine and Phenylalanine metabolism in wheat
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive HF
ORGANISM(S): Triticum Aestivum (ncbitaxon:4565)
SUBMITTER: 
Rainer Schuhmacher
PROVIDER: MSV000097466 | GNPS | Tue Apr 01 04:20:00 BST 2025
REPOSITORIES: GNPS
Similar Datasets
Project description:Part of xx paper will be finished and changed after submission
2025-04-01 | MSV000097466 | MassIVE
Project description:The major Fusarium mycotoxin deoxynivalenol (DON) is a virulence factor in wheat and has also been shown to induce defense responses in host plant tissue. In this study, global and tracer labeling with 13C were combined to annotate the overall metabolome of wheat spikes and to evaluate the response of phenylalanine-related pathways upon treatment with DON. At anthesis, spikes of resistant and susceptible cultivars as well as two related near isogenic wheat lines (NILs) differing in the presence/absence of the major resistance QTL Fhb1 were treated with 1 mg DON or water (control), and samples were collected at 0, 12, 24, 48, and 96 h after treatment (hat). A total of 172 Phe-derived wheat constituents were detected with our untargeted approach employing 13C-labeled phenylalanine and subsequently annotated as flavonoids, lignans, coumarins, benzoic acid derivatives, hydroxycinnamic acid amides (HCAAs), as well as peptides. Ninety-six hours after the DON treatment, up to 30% of the metabolites biosynthesized from Phe showed significantly increased levels compared to the control samples. Major metabolic changes included the formation of precursors of compounds implicated in cell wall reinforcement and presumed antifungal compounds. In addition, also dipeptides, which presumably are products of proteolytic degradation of truncated proteins generated in the presence of the toxin, were significantly more abundant upon DON treatment. An in-depth comparison of the two NILs with correlation clustering of time course profiles revealed some 70 DON-responsive Phe derivatives. While several flavonoids had constitutively different abundance levels between the two NILs differing in resistance, other Phe-derived metabolites such as HCAAs and hydroxycinnamoyl quinates were affected differently in the two NILs after treatment with DON. Our results suggest a strong activation of the general phenylpropanoid pathway and that coumaroyl-CoA is mainly diverted towards HCAAs in the presence of Fhb1, whereas the metabolic route to monolignol(-conjugates), lignans, and lignin seems to be favored in the absence of the Fhb1 resistance quantitative trait loci.
| S-EPMC6831647 | biostudies-literature
Project description:Cancer cells undergo diverse metabolic adaptations to meet the energetic demands imposed by dysregulated growth and proliferation. Assessing metabolism in intact tumors allows the investigator to observe the combined metabolic effects of numerous cancer cell-intrinsic and -extrinsic factors that cannot be fully captured in culture models. We have developed methods to use stable isotope-labeled nutrients (e.g., [13C]glucose) to probe metabolic activity within intact tumors in vivo, in mice and humans. In these methods, the labeled nutrient is introduced to the circulation through an intravenous catheter prior to surgical resection of the tumor and adjacent nonmalignant tissue. Metabolism within these tissues during the infusion transfers the isotope label into metabolic intermediates from pathways supplied by the infused nutrient. Extracting metabolites from surgical specimens and analyzing their isotope labeling patterns provides information about metabolism in the tissue. We provide detailed information about this technique, from introduction of the labeled tracer through data analysis and interpretation, including streamlined approaches to quantify isotope labeling in informative metabolites extracted from tissue samples. We focus on infusions with [13C]glucose and the application of mass spectrometry to assess isotope labeling in intermediates from central metabolic pathways, including glycolysis, the tricarboxylic acid cycle and nonessential amino acid synthesis. We outline practical considerations to apply these methods to human subjects undergoing surgical resections of solid tumors. We also discuss the method's versatility and consider the relative advantages and limitations of alternative approaches to introduce the tracer, harvest the tissue and analyze the data.
| S-EPMC9274147 | biostudies-literature
Project description:Abundant evidence suggests a central role for the amyloid-beta (Aβ) peptide in Alzheimer's disease (AD) pathogenesis. Production and clearance of different Aβ isoforms have been established as targets of proposed disease-modifying therapeutic treatments of AD. However, previous studies used multiple sequential purification steps to isolate the isoforms individually and quantitate them based on a common mid-domain peptide. We created a method to simultaneously purify Aβ isoforms and quantitate them by the specific C-terminal peptides in order to investigate Aβ isoform physiology in the central nervous system. By using standards generated from in vitro metabolic labeling, the relative quantitation of four peptides representing total amount of Aβ (Aβ-Total), Aβ38, Aβ40, and Aβ42 were achieved both in cell culture and in human cerebrospinal fluid (CSF). Standard curves for each isoform demonstrated good sensitivity with very low limits of detection and high accuracy. Because the assay does not require antibody development for each Aβ isoform peptide, significant improvements in the throughput and accuracy of isoform quantitation were achieved.
| S-EPMC3749773 | biostudies-literature
Project description:Xenobiotics are ubiquitous in the environment and modified in the human body by phase I and II metabolism. Liquid chromatography coupled to high resolution mass spectrometry is a powerful tool to investigate these biotransformation products. We present a workflow based on stable isotope-assisted metabolomics and the bioinformatics tool MetExtract II for deciphering xenobiotic metabolites produced by human cells. Its potential was demonstrated by the investigation of the metabolism of deoxynivalenol (DON), an abundant food contaminant, in a liver carcinoma cell line (HepG2) and a model for colon carcinoma (HT29). Detected known metabolites included DON-3-sulfate, DON-10-sulfonate 2, and DON-10-glutathione as well as DON-cysteine. Conjugation with amino acids and an antibiotic was confirmed for the first time. The approach allows the untargeted elucidation of human xenobiotic products in tissue culture. It may be applied to other fields of research including drug metabolism, personalized medicine, exposome research, and systems biology to better understand the relevance of in vitro experiments.
| S-EPMC7171601 | biostudies-literature
Project description:Conspicuous global stable carbon isotope excursions that are recorded in marine sedimentary rocks of Phanerozoic age and were associated with major extinctions have generally paralleled global stable oxygen isotope excursions. All of these phenomena are therefore likely to share a common origin through global climate change. Exceptional patterns for carbon isotope excursions resulted from massive carbon burial during warm intervals of widespread marine anoxic conditions. The many carbon isotope excursions that parallel those for oxygen isotopes can to a large degree be accounted for by the Q10 pattern of respiration for bacteria: As temperature changed along continental margins, where ∼90% of marine carbon burial occurs today, rates of remineralization of isotopically light carbon must have changed exponentially. This would have reduced organic carbon burial during global warming and increased it during global cooling. Also contributing to the δ(13)C excursions have been release and uptake of methane by clathrates, the positive correlation between temperature and degree of fractionation of carbon isotopes by phytoplankton at temperatures below ∼15°, and increased phytoplankton productivity during "icehouse" conditions. The Q10 pattern for bacteria and climate-related changes in clathrate volume represent positive feedbacks for climate change.
| S-EPMC2984178 | biostudies-literature
Project description:We improve on currently-available resources by describing a mass spectrometry (MS)-based strategy using stable isotope dynamic labelling of secretomes (SIDLS) that discriminates between authentic secretory proteins and intracellular proteins within the secretome of cultured cells. By monitoring the rate of incorporation of labelled amino acids into newly synthesised proteins as they appear in the media, we can differentiate those proteins that have been destined for secretion, and exhibit rapid labelling, from those with low rates of labelling or low turnover relative to the growth rate of the cells which is a feature of intracellular proteins. Part of the wet lab protocol for our analysis of secretomes, is the use of the resin, Strataclean (Agilent). In this Supplementary dataset to our main paper (also in PRIDE/ProteomeXchange), we characterise the linearity of capture of secreted proteins using Stracalean resin, by label-free quantitative mass spectrometry. We mix cell-coditioned culture media with 'virgin' media in different ratios and analayse the capture capacity of equal quantitites of StrataClean reagent.
2019-10-08 | PXD009838 | Pride
Project description:Targeted tandem mass spectrometry (LC-MS/MS) has been extremely useful for profiling small molecules extracted from biological sources, such as cells, bodily fluids and tissues. Here, we present a protocol for analysing incorporation of the non-radioactive stable isotopes carbon-13 (13C) and nitrogen-15 (15N) into polar metabolites in central carbon metabolism and related pathways. Our platform utilizes selected reaction monitoring (SRM) with polarity switching and amide hydrophilic interaction liquid chromatography (HILIC) to capture transitions for carbon and nitrogen incorporation into selected metabolites using a hybrid triple quadrupole (QQQ) mass spectrometer. This protocol represents an extension of a previously published protocol for targeted metabolomics of unlabeled species and has been used extensively in tracing the metabolism of nutrients such as 13C-labeled glucose, 13C-glutamine and 15N-glutamine in a variety of biological settings (e.g., cell culture experiments and in vivo mouse labelling via i.p. injection). SRM signals are integrated to produce an array of peak areas for each labelling form that serve as the output for further analysis. The processed data are then used to obtain the degree and distribution of labelling of the targeted molecules (termed fluxomics). Each method can be customized on the basis of known unlabeled Q1/Q3 SRM transitions and adjusted to account for the corresponding 13C or 15N incorporation. The entire procedure takes ~6-7 h for a single sample from experimental labelling and metabolite extraction to peak integration.
| S-EPMC7382369 | biostudies-literature
Project description:Despite the long-established therapeutic efficacy of lithium in the treatment of bipolar disorder (BPD), its molecular mechanism of action remains elusive. Newly developed stable isotope-resolved metabolomics (SIRM) is a powerful approach that can be used to elucidate systematically how lithium impacts glial and neuronal metabolic pathways and activities, leading ultimately to deciphering its molecular mechanism of action. The effect of lithium on the metabolism of three different (13)C-labeled precursors ([U-(13)C]-glucose, (13)C-3-lactate or (13)C-2,3-alanine) was analyzed in cultured rat astrocytes and neurons by nuclear magnetic resonance (NMR) spectroscopy and gas chromatography mass spectrometry (GC-MS). Using [U-(13)C]-glucose, lithium was shown to enhance glycolytic activity and part of the Krebs cycle activity in both astrocytes and neurons, particularly the anaplerotic pyruvate carboxylation (PC). The PC pathway was previously thought to be active in astrocytes but absent in neurons. Lithium also stimulated the extracellular release of (13)C labeled-lactate, -alanine (Ala), -citrate, and -glutamine (Gln) by astrocytes. Interrogation of neuronal pathways using (13)C-3-lactate or (13)C-2,3-Ala as tracers indicated a high capacity of neurons to utilize lactate and Ala in the Krebs cycle, particularly in the production of labeled Asp and Glu via PC and normal cycle activity. Prolonged lithium treatment enhanced lactate metabolism via PC but inhibited lactate oxidation via the normal Krebs cycle in neurons. Such lithium modulation of glycolytic, PC and Krebs cycle activity in astrocytes and neurons as well as release of fuel substrates by astrocytes should help replenish Krebs cycle substrates for Glu synthesis while meeting neuronal demands for energy. Further investigations into the molecular regulation of these metabolic traits should provide new insights into the pathophysiology of mood disorders and early diagnostic markers, as well as new target(s) for effective therapies.
| S-EPMC2903070 | biostudies-literature
Project description:Dehalococcoides species are key players in the anaerobic transformation of halogenated solvents at contaminated sites. Here, we analyze isotopologue distributions in amino acid pools from peptides of Dehalococcoides strain CBDB1 after incubation with (13)C-labeled acetate or bicarbonate as a carbon source. The resulting data were interpreted with regard to genome annotations to identify amino acid biosynthesis pathways. In addition to using gas chromatography-mass spectrometry (GC-MS) for analyzing derivatized amino acids after protein hydrolysis, we introduce a second, much milder method, in which we directly analyze peptide masses after tryptic digest and peptide fragments by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nano-LC-ESI-MS/MS). With this method, we identify isotope incorporation patterns for 17 proteinaceous amino acids, including proline, cysteine, lysine, and arginine, which escaped previous analyses in Dehalococcoides. Our results confirmed lysine biosynthesis via the α-aminoadipate pathway, precluding lysine formation from aspartate. Similarly, the isotopologue pattern obtained for arginine provided biochemical evidence of its synthesis from glutamate. Direct peptide MS/MS analysis of the labeling patterns of glutamine and asparagine, which were converted to glutamate and aspartate during protein hydrolysis, gave biochemical evidence of their precursors and confirmed glutamate biosynthesis via a Re-specific citrate synthase. By addition of unlabeled free amino acids to labeled cells, we show that in strain CBDB1 none of the 17 tested amino acids was incorporated into cell mass, indicating that they are all synthesized de novo. Our approach is widely applicable and provides a means to analyze amino acid metabolism by studying specific proteins even in mixed consortia.
| S-EPMC3416212 | biostudies-literature