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See detailQuantification of Stable Isotope Traces Close to Natural Enrichment in Human Plasma Metabolites Using Gas Chromatography-Mass Spectrometry
Krämer, Lisa; Jäger, Christian UL; Trezzi, Jean-Pierre UL et al

in Metabolites (2018), 8(1), 15

Currently, changes in metabolic fluxes following consumption of stable isotope-enriched foods are usually limited to the analysis of postprandial kinetics of glucose. Kinetic information on a larger ... [more ▼]

Currently, changes in metabolic fluxes following consumption of stable isotope-enriched foods are usually limited to the analysis of postprandial kinetics of glucose. Kinetic information on a larger diversity of metabolites is often lacking, mainly due to the marginal percentage of fully isotopically enriched plant material in the administered food product, and hence, an even weaker 13C enrichment in downstream plasma metabolites. Therefore, we developed an analytical workflow to determine weak 13C enrichments of diverse plasma metabolites with conventional gas chromatography-mass spectrometry (GC-MS). The limit of quantification was increased by optimizing (1) the metabolite extraction from plasma, (2) the GC-MS measurement, and (3) most importantly, the computational data processing. We applied our workflow to study the catabolic dynamics of 13C-enriched wheat bread in three human subjects. For that purpose, we collected time-resolved human plasma samples at 16 timepoints after the consumption of 13C-labeled bread and quantified 13C enrichment of 12 metabolites (glucose, lactate, alanine, glycine, serine, citrate, glutamate, glutamine, valine, isoleucine, tyrosine, and threonine). Based on isotopomer specific analysis, we were able to distinguish catabolic profiles of starch and protein hydrolysis. More generally, our study highlights that conventional GC-MS equipment is sufficient to detect isotope traces below 1% if an appropriate data processing is integrated. [less ▲]

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See detailAnnotating Nontargeted LC-HRMS/MS Data with Two Complementary Tandem Mass Spectral Libraries
Oberacher, Herbert; Reinstadler, Vera; Kreidl, Marco et al

in Metabolites (2018), 9(1 3),

Tandem mass spectral databases are indispensable for fast and reliable compound identification in nontargeted analysis with liquid chromatography–high resolution tandem mass spectrometry (LC-HRMS/MS ... [more ▼]

Tandem mass spectral databases are indispensable for fast and reliable compound identification in nontargeted analysis with liquid chromatography–high resolution tandem mass spectrometry (LC-HRMS/MS), which is applied to a wide range of scientific fields. While many articles now review and compare spectral libraries, in this manuscript we investigate two high-quality and specialized collections from our respective institutes, recorded on different instruments (quadrupole time-of-flight or QqTOF vs. Orbitrap). The optimal range of collision energies for spectral comparison was evaluated using 233 overlapping compounds between the two libraries, revealing that spectra in the range of CE 20–50 eV on the QqTOF and 30–60 nominal collision energy units on the Orbitrap provided optimal matching results for these libraries. Applications to complex samples from the respective institutes revealed that the libraries, combined with a simple data mining approach to retrieve all spectra with precursor and fragment information, could confirm many validated target identifications and yield several new Level 2a (spectral match) identifications. While the results presented are not surprising in many ways, this article adds new results to the debate on the comparability of Orbitrap and QqTOF data and the application of spectral libraries to yield rapid and high-confidence tentative identifications in complex human and environmental samples. [less ▲]

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See detailMind the Gap: Mapping Mass Spectral Databases in Genome-Scale Metabolic Networks Reveals Poorly Covered Areas.
Frainay, Clement; Schymanski, Emma UL; Neumann, Steffen et al

in Metabolites (2018), 8(3),

The use of mass spectrometry-based metabolomics to study human, plant and microbial biochemistry and their interactions with the environment largely depends on the ability to annotate metabolite ... [more ▼]

The use of mass spectrometry-based metabolomics to study human, plant and microbial biochemistry and their interactions with the environment largely depends on the ability to annotate metabolite structures by matching mass spectral features of the measured metabolites to curated spectra of reference standards. While reference databases for metabolomics now provide information for hundreds of thousands of compounds, barely 5% of these known small molecules have experimental data from pure standards. Remarkably, it is still unknown how well existing mass spectral libraries cover the biochemical landscape of prokaryotic and eukaryotic organisms. To address this issue, we have investigated the coverage of 38 genome-scale metabolic networks by public and commercial mass spectral databases, and found that on average only 40% of nodes in metabolic networks could be mapped by mass spectral information from standards. Next, we deciphered computationally which parts of the human metabolic network are poorly covered by mass spectral libraries, revealing gaps in the eicosanoids, vitamins and bile acid metabolism. Finally, our network topology analysis based on the betweenness centrality of metabolites revealed the top 20 most important metabolites that, if added to MS databases, may facilitate human metabolome characterization in the future. [less ▲]

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