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Metabolites
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Sample Preparation in Metabolomics Volume II
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Sample Preparation in Metabolomics Volume II

A special issue of Metabolites (ISSN 2218-1989). This special issue belongs to the section "Metabolomic Profiling Technology".

Deadline for manuscript submissions: closed (20 March 2023) | Viewed by 10903

Special Issue Editors

Prof. Dr. Tommaso Cataldi

E-Mail Website
Guest Editor
Centro Interdipartimentale SMART, Dipartimento di Chimica, Università degli Studi di Bari, Bari, Italy
Interests: LC-MS; metabolic processes; lipids; abiotic stress; neurodegenerative disorders
Dr. Cosima Damiana Calvano

E-Mail Website
Guest Editor
Dipartimento di Chimica, Centro Interdipartimentale SMART, Università degli Studi di Bari, Via Orabona, 4-I-70126 Bari, Italy
Interests: MALDI mass spectrometry; liquid chromatography; food safety; lipidomics; food allergies; food recovery
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

The metabolome has been defined as the qualitative and the quantitative collection of the entire low-molecular-weight molecules, namely metabolites, present in the cells that participate in metabolic reactions and that are essential for their maintenance, growth, and ordinary function. The metabolome includes, among other compounds, amino acids, vitamins, fatty acids, carbohydrates, and lipids.

For metabolite untargeted or targeted profiling aiming at identifying and quantifying a selected number of metabolites, sample preparation plays a critical role to simplify metabolome complexity. This Special Issue of Metabolites will publish reviews and original articles covering the latest developments of sample purification such as solvent precipitation, ultrafiltration, liquid-liquid extraction, and solid-phase extraction for targeted and untargeted analysis using mass spectrometry platforms in application fields such as food analysis, biomedicine, clinical, microbiology,  pharmaceutical and biotechnology industries.

Sample preparation should be the papers’ focus related to deep metabolite coverage, showing the simplicity and minimal handling needed to prevent metabolite loss and/or modification, to reduce the occurrence of extraneous contaminants, and to guarantee reproducibility in metabolome composition.

Finally, comprehensive studies comparing the performance of various sample preparation methods in metabolomics are welcome to aid in the selection of the most appropriate method for a given application.

Prof. Dr. Tommaso Cataldi
Dr. Cosima Damiana Calvano
Guest Editors

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.

 

Keywords

  • metabolome
  • lipids
  • mass spectrometry
  • liquid chromatography
  • solid phase extraction
  • sample handling
  • metabolite extraction
  • purification

Published Papers (4 papers)

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Research

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20 pages, 3289 KiB  
Article
Comparison of Lysis and Detachment Sample Preparation Methods for Cultured Triple-Negative Breast Cancer Cells Using UHPLC–HRMS-Based Metabolomics
by Blake R. Rushing, Madison Schroder and Susan C. J. Sumner
Metabolites 2022, 12(2), 168; https://doi.org/10.3390/metabo12020168 - 10 Feb 2022
Cited by 11 | Viewed by 2452
Abstract
Dysregulation of cellular metabolism is now a well-recognized hallmark of cancer. Studies investigating the metabolic features of cancer cells have shed new light onto processes in cancer cell biology and have identified many potential novel treatment options. The advancement of mass spectrometry-based metabolomics [...] Read more.
Dysregulation of cellular metabolism is now a well-recognized hallmark of cancer. Studies investigating the metabolic features of cancer cells have shed new light onto processes in cancer cell biology and have identified many potential novel treatment options. The advancement of mass spectrometry-based metabolomics has improved the ability to monitor multiple metabolic pathways simultaneously in various experimental settings. However, questions still remain as to how certain steps in the metabolite extraction process affect the metabolic profiles of cancer cells. Here, we use ultra-high-performance liquid chromatography–high-resolution mass spectrometry (UHPLC–HRMS) untargeted metabolomics to investigate the effects of different detachment and lysis methods on the types and abundances of metabolites extracted from MDA-MB-231 cells through the use of in-house standards libraries and pathway analysis software. Results indicate that detachment methods (trypsinization vs. scraping) had the greatest effect on metabolic profiles whereas lysis methods (homogenizer beads vs. freeze–thaw cycling) had a lesser, though still significant, effect. No singular method was clearly superior over others, with certain metabolite classes giving higher abundances or lower variation for each detachment–lysis combination. These results indicate the importance of carefully selecting sample preparation methods for cell-based metabolomics to optimize the extraction performance for certain compound classes. Full article
(This article belongs to the Special Issue Sample Preparation in Metabolomics Volume II)
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19 pages, 5641 KiB  
Article
Fecal 1H-NMR Metabolomics: A Comparison of Sample Preparation Methods for NMR and Novel in Silico Baseline Correction
by Catherine L. J. Brown, Hannah Scott, Crystal Mulik, Amy S. Freund, Michael P. Opyr, Gerlinde A. S. Metz, G. Douglas Inglis and Tony Montina
Metabolites 2022, 12(2), 148; https://doi.org/10.3390/metabo12020148 - 05 Feb 2022
Cited by 6 | Viewed by 2540
Abstract
Analysis of enteric microbiota function indirectly through the fecal metabolome has the potential to be an informative diagnostic tool. However, metabolomic analysis of feces is hampered by high concentrations of macromolecules such as proteins, fats, and fiber in samples. Three methods—ultrafiltration (UF), Bligh–Dyer [...] Read more.
Analysis of enteric microbiota function indirectly through the fecal metabolome has the potential to be an informative diagnostic tool. However, metabolomic analysis of feces is hampered by high concentrations of macromolecules such as proteins, fats, and fiber in samples. Three methods—ultrafiltration (UF), Bligh–Dyer (BD), and no extraction (samples added directly to buffer, vortexed, and centrifuged)—were tested on multiple rat (n = 10) and chicken (n = 8) fecal samples to ascertain whether the methods worked equally well across species and individuals. An in silico baseline correction method was evaluated to determine if an algorithm could produce spectra similar to those obtained via UF. For both rat and chicken feces, UF removed all macromolecules and produced no baseline distortion among samples. By contrast, the BD and no extraction methods did not remove all the macromolecules and produced baseline distortions. The application of in silico baseline correction produced spectra comparable to UF spectra. In the case of no extraction, more intense peaks were produced. This suggests that baseline correction may be a cost-effective method for metabolomic analyses of fecal samples and an alternative to UF. UF was the most versatile and efficient extraction method; however, BD and no extraction followed by baseline correction can produce comparable results. Full article
(This article belongs to the Special Issue Sample Preparation in Metabolomics Volume II)
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19 pages, 1922 KiB  
Article
Automated Sample Preparation and Data Collection Workflow for High-Throughput In Vitro Metabolomics
by Julia M. Malinowska, Taina Palosaari, Jukka Sund, Donatella Carpi, Gavin R. Lloyd, Ralf J. M. Weber, Maurice Whelan and Mark R. Viant
Metabolites 2022, 12(1), 52; https://doi.org/10.3390/metabo12010052 - 08 Jan 2022
Cited by 4 | Viewed by 3528
Abstract
Regulatory bodies have started to recognise the value of in vitro screening and metabolomics as two types of new approach methodologies (NAMs) for chemical risk assessments, yet few high-throughput in vitro toxicometabolomics studies have been reported. A significant challenge is to implement automated [...] Read more.
Regulatory bodies have started to recognise the value of in vitro screening and metabolomics as two types of new approach methodologies (NAMs) for chemical risk assessments, yet few high-throughput in vitro toxicometabolomics studies have been reported. A significant challenge is to implement automated sample preparation of the low biomass samples typically used for in vitro screening. Building on previous work, we have developed, characterised and demonstrated an automated sample preparation and analysis workflow for in vitro metabolomics of HepaRG cells in 96-well microplates using a Biomek i7 Hybrid Workstation (Beckman Coulter) and Orbitrap Elite (Thermo Scientific) high-resolution nanoelectrospray direct infusion mass spectrometry (nESI-DIMS), across polar metabolites and lipids. The experimental conditions evaluated included the day of metabolite extraction, order of extraction of samples in 96-well microplates, position of the 96-well microplate on the instrument’s deck and well location within a microplate. By using the median relative standard deviation (mRSD (%)) of spectral features, we have demonstrated good repeatability of the workflow (final mRSD < 30%) with a low percentage of features outside the threshold applied for statistical analysis. To improve the quality of the automated workflow further, small method modifications were made and then applied to a large cohort study (4860 sample infusions across three nESI-DIMS assays), which confirmed very high repeatability of the whole workflow from cell culturing to metabolite measurements, whilst providing a significant improvement in sample throughput. It is envisioned that the automated in vitro metabolomics workflow will help to advance the application of metabolomics (as a part of NAMs) in chemical safety, primarily as an approach for high throughput screening and prioritisation. Full article
(This article belongs to the Special Issue Sample Preparation in Metabolomics Volume II)
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Review

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12 pages, 1045 KiB  
Review
Isotopic Tracer for Absolute Quantification of Metabolites of the Pentose Phosphate Pathway in Bacteria
by Khairunnisa Mohd Kamal, Mohd Hafidz Mahamad Maifiah, Yan Zhu, Nusaibah Abdul Rahim, Yumi Zuhanis Has-Yun Hashim and Muhamad Shirwan Abdullah Sani
Metabolites 2022, 12(11), 1085; https://doi.org/10.3390/metabo12111085 - 09 Nov 2022
Cited by 2 | Viewed by 1364
Abstract
The pentose phosphate pathway (PPP) plays a key role in many metabolic functions, including the generation of NADPH, biosynthesis of nucleotides, and carbon homeostasis. In particular, the intermediates of PPP have been found to be significantly perturbed in bacterial metabolomic studies. Nonetheless, detailed [...] Read more.
The pentose phosphate pathway (PPP) plays a key role in many metabolic functions, including the generation of NADPH, biosynthesis of nucleotides, and carbon homeostasis. In particular, the intermediates of PPP have been found to be significantly perturbed in bacterial metabolomic studies. Nonetheless, detailed analysis to gain mechanistic information of PPP metabolism remains limited as most studies are unable to report on the absolute levels of the metabolites. Absolute quantification of metabolites is a prerequisite to study the details of fluxes and its regulations. Isotope tracer or labeling studies are conducted in vivo and in vitro and have significantly improved the analysis and understanding of PPP. Due to the laborious procedure and limitations in the in vivo method, an in vitro approach known as Group Specific Internal Standard Technology (GSIST) has been successfully developed to measure the absolute levels of central carbon metabolism, including PPP. The technique adopts derivatization of an experimental sample and a corresponding internal standard with isotope-coded reagents to provide better precision for accurate identification and absolute quantification. In this review, we highlight bacterial studies that employed isotopic tracers as the tagging agents used for the absolute quantification analysis of PPP metabolites. Full article
(This article belongs to the Special Issue Sample Preparation in Metabolomics Volume II)
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