Metabolomics and lipidomics in Caenorhabditis elegans using a single-sample preparation.
Ontology highlight
ABSTRACT:
SUBMITTER: Molenaars M
PROVIDER: S-EPMC8106956 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The goal of this research was to find the most comprehensive lipid extraction of blood plasma, while also providing adequate aqueous preparation for metabolite analysis. Comparisons have been made previously of the Folch, Bligh-Dyer, and Matyash lipid extractions; furthermore, this paper provides an additional comparison of a phospholipid removal plate for analysis. This plate was used for lipid extraction rather than its intended use in lipid removal for polar analysis, and it proves to be robust for targeted lipid analysis. Folch and Matyash provided reproducible recovery over a range of lipid classes, however the Matyash aqueous layer compared well to a typical methanol preparation for polar metabolite analysis. Thus, the Matyash method is the best choice for an untargeted biphasic extraction for metabolomics and lipidomics in blood plasma.
| S-EPMC4582001 | biostudies-literature
Project description:Tumor spheroids are important model systems due to the capability of capturing in vivo tumor complexity. In this work, the experimental design of metabolomics workflows using three-dimensional multicellular tumor spheroid (3D MTS) models is addressed. Non-scaffold based cultures of the HCT116 colon carcinoma cell line delivered highly reproducible MTSs with regard to size and other key parameters (such as protein content and fraction of viable cells) as a prerequisite. Carefully optimizing the multiple steps of sample preparation, the developed procedure enabled us to probe the metabolome of single MTSs (diameter range 790 ± 22 µm) in a highly repeatable manner at a considerable throughput. The final protocol consisted of rapid washing of the spheroids on the cultivation plate, followed by cold methanol extraction. 13C enriched internal standards, added upon extraction, were key to obtaining the excellent analytical figures of merit. Targeted metabolomics provided absolute concentrations with average biological repeatabilities of <20% probing MTSs individually. In a proof of principle study, MTSs were exposed to two metal-based anticancer drugs, oxaliplatin and the investigational anticancer drug KP1339 (sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)]), which exhibit distinctly different modes of action. This difference could be recapitulated in individual metabolic shifts observed from replicate single MTSs. Therefore, biological variation among single spheroids can be assessed using the presented analytical strategy, applicable for in-depth anticancer drug metabolite profiling.
| S-EPMC6950217 | biostudies-literature
Project description:We demonstrate the global metabolic analysis of Caenorhabditis elegans stress responses using a mass-spectrometry-based technique called isotopic ratio outlier analysis (IROA). In an IROA protocol, control and experimental samples are isotopically labeled with 95 and 5% (13)C, and the two sample populations are mixed together for uniform extraction, sample preparation, and LC-MS analysis. This labeling strategy provides several advantages over conventional approaches: (1) compounds arising from biosynthesis are easily distinguished from artifacts, (2) errors from sample extraction and preparation are minimized because the control and experiment are combined into a single sample, (3) measurement of both the molecular weight and the exact number of carbon atoms in each molecule provides extremely accurate molecular formulas, and (4) relative concentrations of all metabolites are easily determined. A heat-shock perturbation was conducted on C. elegans to demonstrate this approach. We identified many compounds that significantly changed upon heat shock, including several from the purine metabolism pathway. The metabolomic response information by IROA may be interpreted in the context of a wealth of genetic and proteomic information available for C. elegans . Furthermore, the IROA protocol can be applied to any organism that can be isotopically labeled, making it a powerful new tool in a global metabolomics pipeline.
| S-EPMC3921967 | biostudies-literature
Project description:Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or identification of new ones can provide invaluable information about the function of a protein of interest. Some of the traditional methods for extract preparation frequently require labor-intensive and time-consuming techniques. Here, a modified extract preparation protocol using a bead mill homogenizer and metal beads is described as a rapid alternative to traditional protein preparation methods. This extract preparation method is compatible with downstream co-immunoprecipitation studies. As an example, the method was used to successfully co-immunoprecipitate C. elegans microRNA Argonaute ALG-1 and two known ALG-1 interactors: AIN-1, and HRPK-1. This protocol includes descriptions of animal sample collection, extract preparation, extract clarification, and protein immunoprecipitation. The described protocol can be adapted to test for interactions between any two or more endogenous, endogenously tagged, or overexpressed C. elegans proteins in a variety of genetic backgrounds.
| S-EPMC7441831 | biostudies-literature
Project description:The identification of lipids in biological samples is gaining importance. The advent of mass spectrometry-based lipidomics accelerated the field allowing nowadays for identification and quantification of complete lipidomes. However, due to solubility difficulties and varying properties of different lipid classes, sample preparation for lipidomics is still an issue. Of the many lipid classes, phospholipids are the major components of biological membranes. In solution, they spontaneously form lipid vesicles of various structures such as liposomes. They are therefore often used as membrane mimics when studying biological membranes and membrane proteins. Here, we present a novel sample preparation strategy for shotgun lipidomics employing liposomes prepared from lipid standards or lipid mixtures allowing the analysis of phospholipids directly from lipid bilayers. We validated our strategy for lipid identification by tandem mass spectrometry in positive or negative ion mode using different phospholipid species from various classes. We further tested our strategy for relative quantification by mixing different ratios of phospholipid species as well as determining the distribution of lipid species in a natural lipid extract. Graphical abstract ᅟ.
| S-EPMC6021460 | biostudies-literature
Project description:Tandem mass spectrometry (MS/MS) with Sequential Window Acquisition of all Theoretical (SWATH) mass spectra generates a comprehensive archive of lipid species within an extract for retrospective, quantitative MS/MS analysis. Here we apply this new technology in Caenorhabditis elegans (C. elegans) to identify potential lipid mediators and pathways. The DAF-1 type I TGF-β and DAF-2 insulin receptors transmit endocrine signals that couple metabolic status to fertility and lifespan. Mutations in daf-1 and daf-2 reduce prostaglandin-endoperoxide synthase (i.e., Cox)-independent prostaglandin synthesis, increase triacylglyceride storage, and alter transcription of numerous lipid metabolism genes. However, the extent to which DAF-1 and DAF-2 signaling modulate lipid metabolism and the underlying mechanisms are not well understood. MS/MSALL with SWATH analysis across the groups identified significant changes in numerous lipids, including specific triacylglycerols, diacylglycerols, and phosphatidylinositols. Examples are provided, using retrospective neutral loss and precursor ion scans as well as MS/MS spectra, to help identify annotated lipids and search libraries for lipids of interest. As proof of principle, we used comparative lipidomics to investigate the prostaglandin metabolism pathway. SWATH data support an unanticipated model: Cox-independent prostaglandin synthesis may involve lysophosphatidylcholine and other lyso glycerophospholipids. This study showcases the power of comprehensive, retrospectively searchable lipid archives as a systems approach for biological discovery in genetic animal models.
| S-EPMC4693190 | biostudies-literature
Project description:Sample pretreatment in conventional cellular metabolomics entails rigorous lysis and extraction steps which increase the duration as well as limit the consistency of these experiments. We report a biomimetic cell culture microfluidic device (MFD) which is coupled with an automated system for rapid, reproducible cell lysis using a combination of electrical and chemical mechanisms. In-channel microelectrodes were created using facile fabrication methods, enabling the application of electric fields up to 1000 V cm(-1). Using this platform, average lysing times were 7.12 s and 3.03 s for chips with no electric fields and electric fields above 200 V cm(-1), respectively. Overall, the electroporation MFDs yielded a ∼10-fold improvement in lysing time over standard chemical approaches. Detection of multiple intracellular nucleotides and energy metabolites in MFD lysates was demonstrated using two different MS platforms. This work will allow for the integrated culture, automated lysis, and metabolic analysis of cells in an MFD which doubles as a biomimetic model of the vasculature.
| S-EPMC4902300 | biostudies-literature
Project description:Lyophilization is a common method used for stabilizing biological samples prior to storage or to concentrate extracts. However, it is possible that this process may alter the metabolic composition or lead to the loss of metabolites. In this study, the performance of lyophilization is investigated in the example of wheat roots. To this end, native and 13C-labelled, fresh or already lyophilized root samples, and (diluted) extracts with dilution factors up to 32 and authentic reference standards were investigated. All samples were analyzed using RP-LC-HRMS. Results show that using lyophilization for the stabilization of plant material altered the metabolic sample composition. Overall, 7% of all wheat metabolites detected in non-lyophilized samples were not detected in dried samples anymore, and up to 43% of the remaining metabolites exhibited significantly increased or decreased abundances. With respect to extract concentration, less than 5% of the expected metabolites were completely lost by lyophilization and the recovery rates of the remaining metabolites were slightly reduced with increasing concentration factors to an average of 85% at an enrichment factor of 32. Compound annotation did not indicate specific classes of wheat metabolites to be affected.
| S-EPMC10300689 | biostudies-literature
Project description:The field of metabolomics generally lacks standardized methods for the preparation of samples prior to analysis. This is especially true for metabolomics of reef-building corals, where the handful of studies that were published employ a range of sample preparation protocols. The utilization of metabolomics may prove essential in understanding coral biology in the face of increasing environmental threats, and an optimized method for preparing coral samples for metabolomics analysis would aid this cause. The current study evaluates three important steps during sample processing of stony corals: (i) metabolite extraction, (ii) metabolism preservation, and (iii) subsampling. Results indicate that a modified Bligh and Dyer extraction is more reproducible across multiple coral species compared to methyl tert-butyl ether and methanol extractions, while a methanol extraction is superior for feature detection. Additionally, few differences were detected between spectra from frozen or lyophilized coral samples. Finally, extraction of entire coral nubbins increased feature detection, but decreased throughput and was more susceptible to subsampling error compared to a novel tissue powder subsampling method. Overall, we recommend the use of a modified Bligh and Dyer extraction, lyophilized samples, and the analysis of brushed tissue powder for the preparation of reef-building coral samples for ¹H NMR metabolomics.
| S-EPMC6410050 | biostudies-literature
Project description:The coupling of transgenes to heat shock promoters is a widely applied method for regulating gene expression. In C. elegans, gene induction can be controlled temporally through timing of heat shock and spatially via targeted rescue in heat shock mutants. Here, we present a method for evoking gene expression in arbitrary cells, with single-cell resolution. We use a focused pulsed infrared laser to locally induce a heat shock response in specific cells. Our method builds on and extends a previously reported method using a continuous-wave laser. In our technique, the pulsed laser illumination enables a much higher degree of spatial selectivity because of diffusion of heat between pulses. We apply our method to induce transient and long-term transgene expression in embryonic, larval, and adult cells. Our method allows highly selective spatiotemporal control of transgene expression and is a powerful tool for model organism biology.
| S-EPMC3789807 | biostudies-other