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Metabolites
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Isotope Guided Metabolomics and Flux Analysis
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Isotope Guided Metabolomics and Flux Analysis

A special issue of Metabolites (ISSN 2218-1989).

Deadline for manuscript submissions: closed (31 October 2017) | Viewed by 28993

Special Issue Editor

Prof. Dr. Wolfgang Eisenreich

E-Mail Website
Guest Editor
Department Chemie, Technische Universität München, Lichtenbergstraße 4, 85747 Garching, Germany
Interests: natural products: isolation, structures, biosynthesis, biological action; bacteria: metabolism, target identification, biotechnology, exploitation; plants: metabolism, terpenoids, biotechnology, exploitation; methods: metabolic profiling, pathway profiling, stable isotopes, target identification
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Labeling experiments using stable isotopes are ma  jor tools in the analysis of biosynthetic and metabolic processes. Moreover, stable isotope labeled metabolites are widely used as internal references to quantify compounds by isotope dilution analysis. Both fields are benefitted by the dramatic progress in mass spectrometry and NMR instrumentation and processing. The Special Issue of Metabolites, "Isotope Guided Metabolomics and Flux Analysis", will be focused on cutting-edge technologies using stable isotopes for metabolic analysis both from a fundamental as well as an applied point of view. The topics that shall be covered by this Special Issue include recent developments and examples in isotope dilution analysis, as well as in isotope guided metabolic pathway and flux analysis. A special focus shall be given to 13C-labeling methods on a global and single cell level, recent developments and applications in metabolic flux analysis, and applications in the analysis of food and pharmaceutical products. Manuscripts dealing with other challenging issues in the field of isotope guided metabolomics and flux analysis are also highly welcome.

Dr. Wolfgang Eisenreich
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Metabolites is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

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Published Papers (5 papers)

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Research

2143 KiB  
Article
Methanol Generates Numerous Artifacts during Sample Extraction and Storage of Extracts in Metabolomics Research
by Claudia Sauerschnig, Maria Doppler, Christoph Bueschl and Rainer Schuhmacher
Metabolites 2018, 8(1), 1; https://doi.org/10.3390/metabo8010001 - 22 Dec 2017
Cited by 46 | Viewed by 7679
Abstract
Many metabolomics studies use mixtures of (acidified) methanol and water for sample extraction. In the present study, we investigated if the extraction with methanol can result in artifacts. To this end, wheat leaves were extracted with mixtures of native and deuterium-labeled methanol and [...] Read more.
Many metabolomics studies use mixtures of (acidified) methanol and water for sample extraction. In the present study, we investigated if the extraction with methanol can result in artifacts. To this end, wheat leaves were extracted with mixtures of native and deuterium-labeled methanol and water, with or without 0.1% formic acid. Subsequently, the extracts were analyzed immediately or after storage at 10 °C, −20 °C or −80 °C with an HPLC-HESI-QExactive HF-Orbitrap instrument. Our results showed that 88 (8%) of the >1100 detected compounds were derived from the reaction with methanol and either formed during sample extraction or short-term storage. Artifacts were found for various substance classes such as flavonoids, carotenoids, tetrapyrrols, fatty acids and other carboxylic acids that are typically investigated in metabolomics studies. 58 of 88 artifacts were common between the two tested extraction variants. Remarkably, 34 of 73 (acidified extraction solvent) and 33 of 73 (non-acidified extraction solvent) artifacts were formed de novo as none of these meth(ox)ylated metabolites were found after extraction of native leaf samples with CD3OH/H2O. Moreover, sample extracts stored at 10 °C for several days, as can typically be the case during longer measurement sequences, led to an increase in both the number and abundance of methylated artifacts. In contrast, frozen sample extracts were relatively stable during a storage period of one week. Our study shows that caution has to be exercised if methanol is used as the extraction solvent as the detected metabolites might be artifacts rather than natural constituents of the biological system. In addition, we recommend storing sample extracts in deep freezers immediately after extraction until measurement. Full article
(This article belongs to the Special Issue Isotope Guided Metabolomics and Flux Analysis)
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4082 KiB  
Article
Dynamics of Monoterpene Formation in Spike Lavender Plants
by Isabel Mendoza-Poudereux, Erika Kutzner, Claudia Huber, Juan Segura, Isabel Arrillaga and Wolfgang Eisenreich
Metabolites 2017, 7(4), 65; https://doi.org/10.3390/metabo7040065 - 19 Dec 2017
Cited by 15 | Viewed by 5861
Abstract
The metabolic cross-talk between the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathways was analyzed in spike lavender (Lavandula latifolia Med) on the basis of 13CO2-labelling experiments using wildtype and transgenic plants overexpressing the 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), the [...] Read more.
The metabolic cross-talk between the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathways was analyzed in spike lavender (Lavandula latifolia Med) on the basis of 13CO2-labelling experiments using wildtype and transgenic plants overexpressing the 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), the first and key enzyme of the MVA pathway. The plants were labelled in the presence of 13CO2 in a gas chamber for controlled pulse and chase periods of time. GC/MS and NMR analysis of 1,8-cineole and camphor, the major monoterpenes present in their essential oil, indicated that the C5-precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) of both monoterpenes are predominantly biosynthesized via the MEP pathway. Surprisingly, overexpression of HMGR did not have significant impact upon the crosstalk between the MVA and MEP pathways indicating that the MEP route is the preferred pathway for the synthesis of C5 monoterpene precursors in spike lavender. Full article
(This article belongs to the Special Issue Isotope Guided Metabolomics and Flux Analysis)
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3196 KiB  
Article
Cell-Type Specific Metabolic Flux Analysis: A Challenge for Metabolic Phenotyping and a Potential Solution in Plants
by Merja T. Rossi, Monika Kalde, Chaiyakorn Srisakvarakul, Nicholas J. Kruger and R. George Ratcliffe
Metabolites 2017, 7(4), 59; https://doi.org/10.3390/metabo7040059 - 13 Nov 2017
Cited by 21 | Viewed by 4587
Abstract
Stable isotope labelling experiments are used routinely in metabolic flux analysis (MFA) to determine the metabolic phenotype of cells and tissues. A complication arises in multicellular systems because single cell measurements of transcriptomes, proteomes and metabolomes in multicellular organisms suggest that the metabolic [...] Read more.
Stable isotope labelling experiments are used routinely in metabolic flux analysis (MFA) to determine the metabolic phenotype of cells and tissues. A complication arises in multicellular systems because single cell measurements of transcriptomes, proteomes and metabolomes in multicellular organisms suggest that the metabolic phenotype will differ between cell types. In silico analysis of simulated metabolite isotopomer datasets shows that cellular heterogeneity confounds conventional MFA because labelling data averaged over multiple cell types does not necessarily yield averaged flux values. A potential solution to this problem—the use of cell-type specific reporter proteins as a source of cell-type specific labelling data—is proposed and the practicality of implementing this strategy in the roots of Arabidopsis thaliana seedlings is explored. A protocol for the immunopurification of ectopically expressed green fluorescent protein (GFP) from Arabidopsis thaliana seedlings using a GFP-binding nanobody is developed, and through GC-MS analysis of protein hydrolysates it is established that constitutively expressed GFP reports accurately on the labelling of total protein in root tissues. It is also demonstrated that the constitutive expression of GFP does not perturb metabolism. The principal obstacle to the implementation of the method in tissues with cell-type specific GFP expression is the sensitivity of the GC-MS system. Full article
(This article belongs to the Special Issue Isotope Guided Metabolomics and Flux Analysis)
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1509 KiB  
Article
Bacterial Substrate Transformation Tracked by Stable-Isotope-Guided NMR Metabolomics: Application in a Natural Aquatic Microbial Community
by Mario Uchimiya, Yuuri Tsuboi, Kengo Ito, Yasuhiro Date and Jun Kikuchi
Metabolites 2017, 7(4), 52; https://doi.org/10.3390/metabo7040052 - 19 Oct 2017
Cited by 8 | Viewed by 4354
Abstract
The transformation of organic substrates by heterotrophic bacteria in aquatic environments constitutes one of the key processes in global material cycles. The development of procedures that would enable us to track the wide range of organic compounds transformed by aquatic bacteria would greatly [...] Read more.
The transformation of organic substrates by heterotrophic bacteria in aquatic environments constitutes one of the key processes in global material cycles. The development of procedures that would enable us to track the wide range of organic compounds transformed by aquatic bacteria would greatly improve our understanding of material cycles. In this study, we examined the applicability of nuclear magnetic resonance spectroscopy coupled with stable-isotope labeling to the investigation of metabolite transformation in a natural aquatic bacterial community. The addition of a model substrate (13C6–glucose) to a coastal seawater sample and subsequent incubation resulted in the detection of >200 peaks and the assignment of 22 metabolites from various chemical classes, including amino acids, dipeptides, organic acids, nucleosides, nucleobases, and amino alcohols, which had been identified as transformed from the 13C6–glucose. Additional experiments revealed large variability in metabolite transformation and the key compounds, showing the bacterial accumulation of glutamate over the incubation period, and that of 3-hydroxybutyrate with increasing concentrations of 13C6–glucose added. These results suggest the potential ability of our approach to track substrate transformation in aquatic bacterial communities. Further applications of this procedure may provide substantial insights into the metabolite dynamics in aquatic environments. Full article
(This article belongs to the Special Issue Isotope Guided Metabolomics and Flux Analysis)
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1314 KiB  
Article
Microscale Quantitative Analysis of Polyhydroxybutyrate in Prokaryotes Using IDMS
by Mariana Itzel Velasco Alvarez, Angela Ten Pierick, Patricia T. N. Van Dam, Reza Maleki Seifar, Mark C. M. Van Loosdrecht and S. Aljoscha Wahl
Metabolites 2017, 7(2), 19; https://doi.org/10.3390/metabo7020019 - 17 May 2017
Cited by 5 | Viewed by 5819
Abstract
Poly(3-hydroxybutyrate) (PHB) is an interesting biopolymer for replacing petroleum-based plastics, its biological production is performed in natural and engineered microorganisms. Current metabolic engineering approaches rely on high-throughput strain construction and screening. Analytical procedures have to be compatible with the small scale and speed [...] Read more.
Poly(3-hydroxybutyrate) (PHB) is an interesting biopolymer for replacing petroleum-based plastics, its biological production is performed in natural and engineered microorganisms. Current metabolic engineering approaches rely on high-throughput strain construction and screening. Analytical procedures have to be compatible with the small scale and speed of these approaches. Here, we present a method based on isotope dilution mass spectrometry (IDMS) and propanolysis extraction of poly(3-hydroxybutyrate) from an Escherichia coli strain engineered for PHB production. As internal standard (IS), we applied an uniformly labeled 13C-cell suspension, of an E. coli PHB producing strain, grown on U-13C-glucose as C-source. This internal 13C-PHB standard enables to quantify low concentrations of PHB (LOD of 0.01 µg/gCDW) from several micrograms of biomass. With this method, a technical reproducibility of about 1.8% relative standard deviation is achieved. Furthermore, the internal standard is robust towards different sample backgrounds and dilutions. The early addition of the internal standard also enables higher reproducibility and increases sensitivity and throughput by simplified sample preparation steps. Full article
(This article belongs to the Special Issue Isotope Guided Metabolomics and Flux Analysis)
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