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17 pages, 1714 KiB

Open AccessArticle

Single-Step Extraction Coupled with Targeted HILIC-MS/MS Approach for Comprehensive Analysis of Human Plasma Lipidome and Polar Metabolome

by Jessica Medina, Vera van der Velpen, Tony Teav, Yann Guitton, Hector Gallart-Ayala and Julijana Ivanisevic

Metabolites 2020, 10(12), 495; https://doi.org/10.3390/metabo10120495 - 2 Dec 2020

Cited by 38 | Viewed by 7661

Abstract

Expanding metabolome coverage to include complex lipids and polar metabolites is essential in the generation of well-founded hypotheses in biological assays. Traditionally, lipid extraction is performed by liquid-liquid extraction using either methyl-tert-butyl ether (MTBE) or chloroform, and polar metabolite extraction using methanol. Here, [...] Read more.

Expanding metabolome coverage to include complex lipids and polar metabolites is essential in the generation of well-founded hypotheses in biological assays. Traditionally, lipid extraction is performed by liquid-liquid extraction using either methyl-tert-butyl ether (MTBE) or chloroform, and polar metabolite extraction using methanol. Here, we evaluated the performance of single-step sample preparation methods for simultaneous extraction of the complex lipidome and polar metabolome from human plasma. The method performance was evaluated using high-coverage Hydrophilic Interaction Liquid Chromatography-ESI coupled to tandem mass spectrometry (HILIC-ESI-MS/MS) methodology targeting a panel of 1159 lipids and 374 polar metabolites. The criteria used for method evaluation comprised protein precipitation efficiency, and relative MS signal abundance and repeatability of detectable lipid and polar metabolites in human plasma. Among the tested methods, the isopropanol (IPA) and 1-butanol:methanol (BUME) mixtures were selected as the best compromises for the simultaneous extraction of complex lipids and polar metabolites, allowing for the detection of 584 lipid species and 116 polar metabolites. The extraction with IPA showed the greatest reproducibility with the highest number of lipid species detected with the coefficient of variation (CV) < 30%. Besides this difference, both IPA and BUME allowed for the high-throughput extraction and reproducible measurement of a large panel of complex lipids and polar metabolites, thus warranting their application in large-scale human population studies. Full article

(This article belongs to the Special Issue Sample Preparation in Metabolomics)

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10 pages, 1617 KiB

Open AccessArticle

General Guidelines for Sample Preparation Strategies in HR-µMAS NMR-based Metabolomics of Microscopic Specimens

by Covadonga Lucas-Torres, Thierry Bernard, Gaspard Huber, Patrick Berthault, Yusuke Nishiyama, Pancham S. Kandiyal, Bénédicte Elena-Herrmann, Laurent Molin, Florence Solari, Anne-Karine Bouzier-Sore and Alan Wong

Metabolites 2020, 10(2), 54; https://doi.org/10.3390/metabo10020054 - 30 Jan 2020

Cited by 11 | Viewed by 4019

Abstract

The study of the metabolome within tissues, organisms, cells or biofluids can be carried out by several bioanalytical techniques. Among them, nuclear magnetic resonance (NMR) is one of the principal spectroscopic methods. This is due to a sample rotation technique, high-resolution magic angle [...] Read more.

The study of the metabolome within tissues, organisms, cells or biofluids can be carried out by several bioanalytical techniques. Among them, nuclear magnetic resonance (NMR) is one of the principal spectroscopic methods. This is due to a sample rotation technique, high-resolution magic angle spinning (HR-MAS), which targets the analysis of heterogeneous specimens with a bulk sample mass from 5 to 10 mg. Recently, a new approach, high-resolution micro-magic angle spinning (HR-μMAS), has been introduced. It opens, for the first time, the possibility of investigating microscopic specimens (<500 μg) with NMR spectroscopy, strengthening the concept of homogeneous sampling in a heterogeneous specimen. As in all bioanalytical approaches, a clean and reliable sample preparation strategy is a significant component in designing metabolomics (or -omics, in general) studies. The sample preparation for HR-μMAS is consequentially complicated by the μg-scale specimen and has yet to be addressed. This report details the strategies for three specimen types: biofluids, fluid matrices and tissues. It also provides the basis for designing future μMAS NMR studies of microscopic specimens. Full article

(This article belongs to the Special Issue Sample Preparation in Metabolomics)

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17 pages, 2934 KiB

Open AccessArticle

Electromembrane Extraction of Highly Polar Compounds: Analysis of Cardiovascular Biomarkers in Plasma

by Nicolas Drouin, Tim Kloots, Julie Schappler, Serge Rudaz, Isabelle Kohler, Amy Harms, Petrus Wilhelmus Lindenburg and Thomas Hankemeier

Metabolites 2020, 10(1), 4; https://doi.org/10.3390/metabo10010004 - 18 Dec 2019

Cited by 26 | Viewed by 4060

Abstract

Cardiovascular diseases (CVDs) represent a major concern in today’s society, with more than 17.5 million deaths reported annually worldwide. Recently, five metabolites related to the gut metabolism of phospholipids were identified as promising predictive biomarker candidates for CVD. Validation of those biomarker candidates [...] Read more.

Cardiovascular diseases (CVDs) represent a major concern in today’s society, with more than 17.5 million deaths reported annually worldwide. Recently, five metabolites related to the gut metabolism of phospholipids were identified as promising predictive biomarker candidates for CVD. Validation of those biomarker candidates is crucial for applications to the clinic, showing the need for high-throughput analysis of large numbers of samples. These five compounds, trimethylamine N-oxide (TMAO), choline, betaine, l-carnitine, and deoxy-l-carnitine (4-trimethylammoniobutanoic acid), are highly polar compounds and show poor retention on conventional reversed phase chromatography, which can lead to strong matrix effects when using mass spectrometry detection, especially when high-throughput analysis approaches are used with limited separation of analytes from interferences. In order to reduce the potential matrix effects, we propose a novel fast parallel electromembrane extraction (Pa-EME) method for the analysis of these metabolites in plasma samples. The evaluation of Pa-EME parameters was performed using multi segment injection–capillary electrophoresis–mass spectrometry (MSI-CE-MS). Recoveries up to 100% were achieved, with variability as low as 2%. Overall, this study highlights the necessity of protein precipitation prior to EME for the extraction of highly polar compounds. The developed Pa-EME method was evaluated in terms of concentration range and response function, as well as matrix effects using fast-LC-MS/MS. Finally, the developed workflow was compared to conventional sample pre-treatment, i.e., protein precipitation using methanol, and fast-LC-MS/MS. Data show very strong correlations between both workflows, highlighting the great potential of Pa-EME for high-throughput biological applications. Full article

(This article belongs to the Special Issue Sample Preparation in Metabolomics)

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14 pages, 2703 KiB

Open AccessArticle

Modified Protocol of Harvesting, Extraction, and Normalization Approaches for Gas Chromatography Mass Spectrometry-Based Metabolomics Analysis of Adherent Cells Grown Under High Fetal Calf Serum Conditions

by Raphaela Fritsche-Guenther, Anna Bauer, Yoann Gloaguen, Mario Lorenz and Jennifer A. Kirwan

Metabolites 2020, 10(1), 2; https://doi.org/10.3390/metabo10010002 - 18 Dec 2019

Cited by 8 | Viewed by 4724

Abstract

A gas chromatography mass spectrometry (GC-MS) metabolomics protocol was modified for quenching, harvesting, and extraction of metabolites from adherent cells grown under high (20%) fetal calf serum conditions. The reproducibility of using either 50% or 80% methanol for quenching of cells was compared [...] Read more.

A gas chromatography mass spectrometry (GC-MS) metabolomics protocol was modified for quenching, harvesting, and extraction of metabolites from adherent cells grown under high (20%) fetal calf serum conditions. The reproducibility of using either 50% or 80% methanol for quenching of cells was compared for sample harvest. To investigate the efficiency and reproducibility of intracellular metabolite extraction, different volumes and ratios of chloroform were tested. Additionally, we compared the use of total protein amount versus cell mass as normalization parameters. We demonstrate that the method involving 50% methanol as quenching buffer followed by an extraction step using an equal ratio of methanol:chloroform:water (1:1:1, v/v/v) followed by the collection of 6 mL polar phase for GC-MS measurement was superior to the other methods tested. Especially for large sample sets, its comparative ease of measurement leads us to recommend normalization to protein amount for the investigation of intracellular metabolites of adherent human cells grown under high (or standard) fetal calf serum conditions. To avoid bias, care should be taken beforehand to ensure that the ratio of total protein to cell number are consistent among the groups tested. For this reason, it may not be suitable where culture conditions or cell types have very different protein outputs (e.g., hypoxia vs. normoxia). The full modified protocol is available in the Supplementary Materials. Full article

(This article belongs to the Special Issue Sample Preparation in Metabolomics)

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21 pages, 2854 KiB

Open AccessArticle

Single Spheroid Metabolomics: Optimizing Sample Preparation of Three-Dimensional Multicellular Tumor Spheroids

by Mate Rusz, Evelyn Rampler, Bernhard K. Keppler, Michael A. Jakupec and Gunda Koellensperger

Metabolites 2019, 9(12), 304; https://doi.org/10.3390/metabo9120304 - 14 Dec 2019

Cited by 19 | Viewed by 4450

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Sample Preparation in Metabolomics)

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16 pages, 1671 KiB

Open AccessArticle

Influence of Drying Method on NMR-Based Metabolic Profiling of Human Cell Lines

by Irina Petrova, Shenyuan Xu, William C. Joesten, Shuisong Ni and Michael A. Kennedy

Metabolites 2019, 9(11), 256; https://doi.org/10.3390/metabo9110256 - 31 Oct 2019

Cited by 7 | Viewed by 3164

Abstract

Metabolic profiling of cell line and tissue extracts involves sample processing that includes a drying step prior to re-dissolving the cell or tissue extracts in a buffer for analysis by GC/LC-MS or NMR. Two of the most commonly used drying techniques are centrifugal [...] Read more.

Metabolic profiling of cell line and tissue extracts involves sample processing that includes a drying step prior to re-dissolving the cell or tissue extracts in a buffer for analysis by GC/LC-MS or NMR. Two of the most commonly used drying techniques are centrifugal evaporation under vacuum (SpeedVac) and lyophilization. Here, NMR spectroscopy was used to determine how the metabolic profiles of hydrophilic extracts of three human pancreatic cancer cell lines, MiaPaCa-2, Panc-1 and AsPC-1, were influenced by the choice of drying technique. In each of the three cell lines, 40–50 metabolites were identified as having statistically significant differences in abundance in redissolved extract samples depending on the drying technique used during sample preparation. In addition to these differences, some metabolites were only present in the lyophilized samples, for example, n-methyl-α-aminoisobutyric acid, n-methylnicotimamide, sarcosine and 3-hydroxyisovaleric acid, whereas some metabolites were only present in SpeedVac dried samples, for example, trimethylamine. This research demonstrates that the choice of drying technique used during the preparation of samples of human cell lines or tissue extracts can significantly influence the observed metabolome, making it important to carefully consider the selection of a drying method prior to preparation of such samples for metabolic profiling. Full article

(This article belongs to the Special Issue Sample Preparation in Metabolomics)

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15 pages, 1835 KiB

Open AccessArticle

Choosing an Optimal Sample Preparation in Caulobacter crescentus for Untargeted Metabolomics Approaches

by Julian Pezzatti, Matthieu Bergé, Julien Boccard, Santiago Codesido, Yoric Gagnebin, Patrick H. Viollier, Víctor González-Ruiz and Serge Rudaz

Metabolites 2019, 9(10), 193; https://doi.org/10.3390/metabo9100193 - 20 Sep 2019

Cited by 10 | Viewed by 3458

Abstract

Untargeted metabolomics aims to provide a global picture of the metabolites present in the system under study. To this end, making a careful choice of sample preparation is mandatory to obtain reliable and reproducible biological information. In this study, eight different sample preparation [...] Read more.

Untargeted metabolomics aims to provide a global picture of the metabolites present in the system under study. To this end, making a careful choice of sample preparation is mandatory to obtain reliable and reproducible biological information. In this study, eight different sample preparation techniques were evaluated using Caulobacter crescentus as a model for Gram-negative bacteria. Two cell retrieval systems, two quenching and extraction solvents, and two cell disruption procedures were combined in a full factorial experimental design. To fully exploit the multivariate structure of the generated data, the ANOVA multiblock orthogonal partial least squares (AMOPLS) algorithm was employed to decompose the contribution of each factor studied and their potential interactions for a set of annotated metabolites. All main effects of the factors studied were found to have a significant contribution on the total observed variability. Cell retrieval, quenching and extraction solvent, and cell disrupting mechanism accounted respectively for 27.6%, 8.4%, and 7.0% of the total variability. The reproducibility and metabolome coverage of the sample preparation procedures were then compared and evaluated in terms of relative standard deviation (RSD) on the area for the detected metabolites. The protocol showing the best performance in terms of recovery, versatility, and variability was centrifugation for cell retrieval, using MeOH:H₂O (8:2) as quenching and extraction solvent, and freeze-thaw cycles as the cell disrupting mechanism. Full article

(This article belongs to the Special Issue Sample Preparation in Metabolomics)

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Review

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17 pages, 1741 KiB

Open AccessReview

Current Practice in Untargeted Human Milk Metabolomics

by Isabel Ten-Doménech, Victoria Ramos-Garcia, José David Piñeiro-Ramos, María Gormaz, Anna Parra-Llorca, Máximo Vento, Julia Kuligowski and Guillermo Quintás

Metabolites 2020, 10(2), 43; https://doi.org/10.3390/metabo10020043 - 22 Jan 2020

Cited by 31 | Viewed by 5466

Abstract

Human milk (HM) is considered the gold standard for infant nutrition. HM contains macro- and micronutrients, as well as a range of bioactive compounds (hormones, growth factors, cell debris, etc.). The analysis of the complex and dynamic composition of HM has been a [...] Read more.

Human milk (HM) is considered the gold standard for infant nutrition. HM contains macro- and micronutrients, as well as a range of bioactive compounds (hormones, growth factors, cell debris, etc.). The analysis of the complex and dynamic composition of HM has been a permanent challenge for researchers. The use of novel, cutting-edge techniques involving different metabolomics platforms has permitted to expand knowledge on the variable composition of HM. This review aims to present the state-of-the-art in untargeted metabolomic studies of HM, with emphasis on sampling, extraction and analysis steps. Workflows available from the literature have been critically revised and compared, including a comprehensive assessment of the achievable metabolome coverage. Based on the scientific evidence available, recommendations for future untargeted HM metabolomics studies are included. Full article

(This article belongs to the Special Issue Sample Preparation in Metabolomics)

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30 pages, 917 KiB

Open AccessEditor’s ChoiceReview

Metabolomics in the Context of Plant Natural Products Research: From Sample Preparation to Metabolite Analysis

by Mohamed A. Salem, Leonardo Perez de Souza, Ahmed Serag, Alisdair R. Fernie, Mohamed A. Farag, Shahira M. Ezzat and Saleh Alseekh

Metabolites 2020, 10(1), 37; https://doi.org/10.3390/metabo10010037 - 15 Jan 2020

Cited by 194 | Viewed by 15924

Abstract

Plant-derived natural products have long been considered a valuable source of lead compounds for drug development. Natural extracts are usually composed of hundreds to thousands of metabolites, whereby the bioactivity of natural extracts can be represented by synergism between several metabolites. However, isolating [...] Read more.

Plant-derived natural products have long been considered a valuable source of lead compounds for drug development. Natural extracts are usually composed of hundreds to thousands of metabolites, whereby the bioactivity of natural extracts can be represented by synergism between several metabolites. However, isolating every single compound from a natural extract is not always possible due to the complex chemistry and presence of most secondary metabolites at very low levels. Metabolomics has emerged in recent years as an indispensable tool for the analysis of thousands of metabolites from crude natural extracts, leading to a paradigm shift in natural products drug research. Analytical methods such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) are used to comprehensively annotate the constituents of plant natural products for screening, drug discovery as well as for quality control purposes such as those required for phytomedicine. In this review, the current advancements in plant sample preparation, sample measurements, and data analysis are presented alongside a few case studies of the successful applications of these processes in plant natural product drug discovery. Full article

(This article belongs to the Special Issue Sample Preparation in Metabolomics)

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16 pages, 472 KiB

Open AccessReview

Experimental Design and Sample Preparation in Forest Tree Metabolomics

by Ana M. Rodrigues, Ana I. Ribeiro-Barros and Carla António

Metabolites 2019, 9(12), 285; https://doi.org/10.3390/metabo9120285 - 22 Nov 2019

Cited by 30 | Viewed by 6028

Abstract

Appropriate experimental design and sample preparation are key steps in metabolomics experiments, highly influencing the biological interpretation of the results. The sample preparation workflow for plant metabolomics studies includes several steps before metabolite extraction and analysis. These include the optimization of laboratory procedures, [...] Read more.

Appropriate experimental design and sample preparation are key steps in metabolomics experiments, highly influencing the biological interpretation of the results. The sample preparation workflow for plant metabolomics studies includes several steps before metabolite extraction and analysis. These include the optimization of laboratory procedures, which should be optimized for different plants and tissues. This is particularly the case for trees, whose tissues are complex matrices to work with due to the presence of several interferents, such as oleoresins, cellulose. A good experimental design, tree tissue harvest conditions, and sample preparation are crucial to ensure consistency and reproducibility of the metadata among datasets. In this review, we discuss the main challenges when setting up a forest tree metabolomics experiment for mass spectrometry (MS)-based analysis covering all technical aspects from the biological question formulation and experimental design to sample processing and metabolite extraction and data acquisition. We also highlight the importance of forest tree metadata standardization in metabolomics studies. Full article

(This article belongs to the Special Issue Sample Preparation in Metabolomics)

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14 pages, 443 KiB

Open AccessReview

Metabolomics Applied to the Study of Extracellular Vesicles

by Charles Williams, Mari Palviainen, Niels-Christian Reichardt, Pia R.-M. Siljander and Juan M. Falcón-Pérez

Metabolites 2019, 9(11), 276; https://doi.org/10.3390/metabo9110276 - 12 Nov 2019

Cited by 71 | Viewed by 7262

Abstract

Cell-secreted extracellular vesicles (EVs) have rapidly gained prominence as sources of biomarkers for non-invasive biopsies, owing to their ubiquity across human biofluids and physiological stability. There are many characterisation studies directed towards their protein, nucleic acid, lipid and glycan content, but more recently [...] Read more.

Cell-secreted extracellular vesicles (EVs) have rapidly gained prominence as sources of biomarkers for non-invasive biopsies, owing to their ubiquity across human biofluids and physiological stability. There are many characterisation studies directed towards their protein, nucleic acid, lipid and glycan content, but more recently the metabolomic analysis of EV content has also gained traction. Several EV metabolite biomarker candidates have been identified across a range of diseases, including liver disease and cancers of the prostate and pancreas. Beyond clinical applications, metabolomics has also elucidated possible mechanisms of action underlying EV function, such as the arginase-mediated relaxation of pulmonary arteries or the delivery of nutrients to tumours by vesicles. However, whilst the value of EV metabolomics is clear, there are challenges inherent to working with these entities—particularly in relation to sample production and preparation. The biomolecular composition of EVs is known to change drastically depending on the isolation method used, and recent evidence has demonstrated that changes in cell culture systems impact upon the metabolome of the resulting EVs. This review aims to collect recent advances in the EV metabolomics field whilst also introducing researchers interested in this area to practical pitfalls in applying metabolomics to EV studies. Full article

(This article belongs to the Special Issue Sample Preparation in Metabolomics)

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