Elemental formula annotation of polar and lipophilic metabolites using 13C, 15N and 34S isotope labelling, in combination with high-resolution mass spectrometry
TL;DR: A comprehensive multi-isotope labelling-based strategy using fully labelled plant tissues, in combination with a fractionated metabolite extraction protocol, which can be applied in either an automated database-dependent or a database-independent analysis of the plant polar metabolome and lipidome.
Abstract: The unbiased and comprehensive analysis of metabolites in any organism presents a major challenge if proper peak annotation and unambiguous assignment of the biological origin of the peaks are required. Here we provide a comprehensive multi-isotope labelling-based strategy using fully labelled (13) C, (15) N and (34) S plant tissues, in combination with a fractionated metabolite extraction protocol. The extraction procedure allows for the simultaneous extraction of polar, semi-polar and hydrophobic metabolites, as well as for the extraction of proteins and starch. After labelling and extraction, the metabolites and lipids were analysed using a high-resolution mass spectrometer providing accurate MS and all-ion fragmentation data, providing an unambiguous readout for every detectable isotope-labelled peak. The isotope labelling assisted peak annotation process employed can be applied in either an automated database-dependent or a database-independent analysis of the plant polar metabolome and lipidome. As a proof of concept, the developed methods and technologies were applied and validated using Arabidopsis thaliana leaf and root extracts. Along with a large repository of assigned elemental compositions, which is provided, we show, using selected examples, the accuracy and reliability of the developed workflow.
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TL;DR: This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids that represent their major form of carbon and energy storage in Arabidopsis.
Abstract: Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables.
1,169 citations
Cites background from "Elemental formula annotation of pol..."
...Extraction of lipids with methanol:tert-butyl-ether:water (1:3:1) has been recommended if quantitative recovery of proteins and starch from the extract is desirable (Giavalisco et al., 2011)....
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TL;DR: This work focuses on the application of mass spectrometry to the treatment of metabolomics and Lipidomics with a focus on the characterization of the role of phosphorous in the biosynthetic pathway.
Abstract: in Mass Spectrometry-Based Metabolomics and Lipidomics Tomas Cajka† and Oliver Fiehn*,†,‡ †UC Davis Genome Center−Metabolomics, University of California Davis, 451 Health Sciences Drive, Davis, California 95616, United States ‡King Abdulaziz University, Faculty of Science, Biochemistry Department, P.O. Box 80203, Jeddah 21589, Saudi Arabia ■ CONTENTS Sample Extraction 525 Extraction of Polar Metabolites (Metabolomics) 525 Extraction of Lipids (Lipidomics) 527 Combined Extraction of Amphiphilic and Lipophilic Metabolites 527 Mass Spectrometry-Based Metabolomics and Lipidomics 528 Direct Infusion MS 528 Ion Mobility-Mass Spectrometry (IM-MS) 529 Liquid Chromatography−Mass Spectrometry (LC−MS) 533 Reversed-Phase Liquid Chromatography (RPLC) 533 Hydrophilic Interaction Chromatography (HILIC) 534 Normal-Phase Liquid Chromatography (NPLC) 535 Supercritical Fluid Chromatography (SFC) 535 Two-Dimensional Liquid Chromatography (2D-LC) 535 Mass Spectrometric Detection 536 Data Processing 540 Quality Control 541 Conclusions 541 Author Information 542 Corresponding Author 542 Notes 542 Biographies 542 Acknowledgments 542 References 542
549 citations
TL;DR: This review focuses on state of the art in LC-MS-based lipidomics, covering all the steps of global lipidomic profiling, and concludes that phospholipids followed by sphingomyelins, di- and tri-acylglycerols, and ceramides were the most frequently targeted lipid species.
Abstract: Liquid chromatography-mass spectrometry (LC-MS)-based lipidomics has undergone dramatic developments over the past decade. This review focuses on state of the art in LC-MS-based lipidomics, covering all the steps of global lipidomic profiling. By reviewing 185 original papers and application notes, we can conclude that current advanced LC-MS-based lipidomics methods involve: (1) lipid extraction schemes using chloroform/MeOH or methyl tert -butyl ether (MTBE)/MeOH, both with addition of internal standards covering each lipid class; (2) LC separation of lipids using short microbore C18 or C8 columns with sub-2-µm or 2.6–2.8-µm (fused-core) particle size with analysis time (3) electrospray ionization in positive- and negative-ion modes with full spectra acquisition using high-resolution MS with capability to MS/MS. Phospholipids (phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, phosphatidylserines, phosphatidylglycerols) followed by sphingomyelins, di- and tri-acylglycerols, and ceramides were the most frequently targeted lipid species.
460 citations
TL;DR: The metabolome data and the approach provided here can serve as a blueprint for the analysis of traits and conditions linking crop yield and senescence as well as provide clues to source-sink relations.
Abstract: Developmental senescence is a coordinated physiological process in plants and is critical for nutrient redistribution from senescing leaves to newly formed sink organs, including young leaves and developing seeds. Progress has been made concerning the genes involved and the regulatory networks controlling senescence. The resulting complex metabolome changes during senescence have not been investigated in detail yet. Therefore, we conducted a comprehensive profiling of metabolites, including pigments, lipids, sugars, amino acids, organic acids, nutrient ions, and secondary metabolites, and determined approximately 260 metabolites at distinct stages in leaves and siliques during senescence in Arabidopsis (Arabidopsis thaliana). This provided an extensive catalog of metabolites and their spatiotemporal cobehavior with progressing senescence. Comparison with silique data provides clues to source-sink relations. Furthermore, we analyzed the metabolite distribution within single leaves along the basipetal sink-source transition trajectory during senescence. Ceramides, lysolipids, aromatic amino acids, branched chain amino acids, and stress-induced amino acids accumulated, and an imbalance of asparagine/aspartate, glutamate/glutamine, and nutrient ions in the tip region of leaves was detected. Furthermore, the spatiotemporal distribution of tricarboxylic acid cycle intermediates was already changed in the presenescent leaves, and glucosinolates, raffinose, and galactinol accumulated in the base region of leaves with preceding senescence. These results are discussed in the context of current models of the metabolic shifts occurring during developmental and environmentally induced senescence. As senescence processes are correlated to crop yield, the metabolome data and the approach provided here can serve as a blueprint for the analysis of traits and conditions linking crop yield and senescence.
254 citations
Cites methods from "Elemental formula annotation of pol..."
...Frozen ground material (50 mg) was homogenized as described previously (Giavalisco et al., 2011; Hummel et al., 2011) in 1 mL of a cold 1:3 (v/v) methanol:methyl-tert-butyl-ether solution that was subjected to constant shaking and sonication....
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...…changes, including the onset of metabolite remobilization, during developmental senescence in rosette leaves, upper leaves, and siliques, profiling of lipophilic metabolites by ultraperformance liquid chromatography (UPLC)-mass spectrometry (MS; Giavalisco et al., 2011), primary Fresh weight....
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...UPLC separations were subsequently performed as described (Giavalisco et al., 2011; Hummel et al., 2011) on a Waters Acquity UPLC system equipped with a BEH C8 reversephase column (Waters) coupled to an Exactive Orbitrap (Thermo Fisher Scientific)....
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TL;DR: Recent applications of metabolomics are described and its increasing application to study plant responses to environmental (stress-) factors, including drought, salt, low oxygen caused by waterlogging or flooding of the soil, temperature, light and oxidative stress are described.
Abstract: Metabolomics is one omics approach that can be used to acquire comprehensive information on the composition of a metabolite pool to provide a functional screen of the cellular state. Studies of the plant metabolome include analysis of a wide range of chemical species with diverse physical properties, from ionic inorganic compounds to biochemically derived hydrophilic carbohydrates, organic and amino acids, and a range of hydrophobic lipid-related compounds. This complexitiy brings huge challenges to the analytical technologies employed in current plant metabolomics programs, and powerful analytical tools are required for the separation and characterization of this extremely high compound diversity present in biological sample matrices. The use of mass spectrometry (MS)-based analytical platforms to profile stress-responsive metabolites that allow some plants to adapt to adverse environmental conditions is fundamental in current plant biotechnology research programs for the understanding and development of stress-tolerant plants. In this review, we describe recent applications of metabolomics and emphasize its increasing application to study plant responses to environmental (stress-) factors, including drought, salt, low oxygen caused by waterlogging or flooding of the soil, temperature, light and oxidative stress (or a combination of them). Advances in understanding the global changes occurring in plant metabolism under specific abiotic stress conditions are fundamental to enhance plant fitness and increase stress tolerance. © 2015 Wiley Periodicals, Inc. Mass Spec Rev 35:620-649, 2016.
243 citations
Cites methods from "Elemental formula annotation of pol..."
...For example, LC–FT–ICR–MS was used together with stable isotopes (e.g., 13C and 15N) to label metabolites, to improve the quality of plant metabolite annotation (Giavalisco et al., 2009, 2011)....
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References
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TL;DR: KEGG PATHWAY is now supplemented with a new global map of metabolic pathways, which is essentially a combined map of about 120 existing pathway maps, and the KEGG resource is being expanded to suit the needs for practical applications.
Abstract: KEGG (http://www.genome.jp/kegg/) is a database of biological systems that integrates genomic, chemical and systemic functional information. KEGG provides a reference knowledge base for linking genomes to life through the process of PATHWAY mapping, which is to map, for example, a genomic or transcriptomic content of genes to KEGG reference pathways to infer systemic behaviors of the cell or the organism. In addition, KEGG provides a reference knowledge base for linking genomes to the environment, such as for the analysis of drug-target relationships, through the process of BRITE mapping. KEGG BRITE is an ontology database representing functional hierarchies of various biological objects, including molecules, cells, organisms, diseases and drugs, as well as relationships among them. KEGG PATHWAY is now supplemented with a new global map of metabolic pathways, which is essentially a combined map of about 120 existing pathway maps. In addition, smaller pathway modules are defined and stored in KEGG MODULE that also contains other functional units and complexes. The KEGG resource is being expanded to suit the needs for practical applications. KEGG DRUG contains all approved drugs in the US and Japan, and KEGG DISEASE is a new database linking disease genes, pathways, drugs and diagnostic markers.
5,352 citations
TL;DR: An LC/MS-based data analysis approach, XCMS, which incorporates novel nonlinear retention time alignment, matched filtration, peak detection, and peak matching, and is demonstrated using data sets from a previously reported enzyme knockout study and a large-scale study of plasma samples.
Abstract: Metabolite profiling in biomarker discovery, enzyme substrate assignment, drug activity/specificity determination, and basic metabolic research requires new data preprocessing approaches to correlate specific metabolites to their biological origin. Here we introduce an LC/MS-based data analysis approach, XCMS, which incorporates novel nonlinear retention time alignment, matched filtration, peak detection, and peak matching. Without using internal standards, the method dynamically identifies hundreds of endogenous metabolites for use as standards, calculating a nonlinear retention time correction profile for each sample. Following retention time correction, the relative metabolite ion intensities are directly compared to identify changes in specific endogenous metabolites, such as potential biomarkers. The software is demonstrated using data sets from a previously reported enzyme knockout study and a large-scale study of plasma samples. XCMS is freely available under an open-source license at http://metlin...
3,963 citations
Additional excerpts
...…improvements in open-source and commercial software developments, allowing data extraction and initial processing (Broeckling et al., 2006; Smith et al., 2006; Katajamaa and Oresic, 2007; Lommen, 2009; Benton et al., 2010; Pluskal et al., 2010), and the increasing quality and…...
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TL;DR: A new generation of a popular open-source data processing toolbox, MZmine 2 is introduced, suitable for processing large batches of data and has been applied to both targeted and non-targeted metabolomic analyses.
Abstract: Mass spectrometry (MS) coupled with online separation methods is commonly applied for differential and quantitative profiling of biological samples in metabolomic as well as proteomic research. Such approaches are used for systems biology, functional genomics, and biomarker discovery, among others. An ongoing challenge of these molecular profiling approaches, however, is the development of better data processing methods. Here we introduce a new generation of a popular open-source data processing toolbox, MZmine 2. A key concept of the MZmine 2 software design is the strict separation of core functionality and data processing modules, with emphasis on easy usability and support for high-resolution spectra processing. Data processing modules take advantage of embedded visualization tools, allowing for immediate previews of parameter settings. Newly introduced functionality includes the identification of peaks using online databases, MSn data support, improved isotope pattern support, scatter plot visualization, and a new method for peak list alignment based on the random sample consensus (RANSAC) algorithm. The performance of the RANSAC alignment was evaluated using synthetic datasets as well as actual experimental data, and the results were compared to those obtained using other alignment algorithms. MZmine 2 is freely available under a GNU GPL license and can be obtained from the project website at: http://mzmine.sourceforge.net/
. The current version of MZmine 2 is suitable for processing large batches of data and has been applied to both targeted and non-targeted metabolomic analyses.
2,884 citations
"Elemental formula annotation of pol..." refers background in this paper
...…data extraction and initial processing (Broeckling et al., 2006; Smith et al., 2006; Katajamaa and Oresic, 2007; Lommen, 2009; Benton et al., 2010; Pluskal et al., 2010), and the increasing quality and accessibility of public databases (Williams, 2008; Tohge and Fernie, 2009; Wang et al., 2009)....
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TL;DR: This review presents an overview of the dynamically developing field of mass spectrometry-based metabolomics, a technique that analyzes all detectable analytes in a given sample with subsequent classification of samples and identification of differentially expressed metabolites, which define the sample classes.
Abstract: This review presents an overview of the dynamically developing field of mass spectrometry-based metabolomics. Metabolomics aims at the comprehensive and quantitative analysis of wide arrays of metabolites in biological samples. These numerous analytes have very diverse physico-chemical properties and occur at different abundance levels. Consequently, comprehensive metabolomics investigations are primarily a challenge for analytical chemistry and specifically mass spectrometry has vast potential as a tool for this type of investigation. Metabolomics require special approaches for sample preparation, separation, and mass spectrometric analysis. Current examples of those approaches are described in this review. It primarily focuses on metabolic fingerprinting, a technique that analyzes all detectable analytes in a given sample with subsequent classification of samples and identification of differentially expressed metabolites, which define the sample classes. To perform this complex task, data analysis tools, metabolite libraries, and databases are required. Therefore, recent advances in metabolomics bioinformatics are also discussed.
1,954 citations
"Elemental formula annotation of pol..." refers background or methods in this paper
...NMR-based analysis (Dettmer et al., 2007; Eisenreich and Bacher, 2007; Pan and Raftery, 2007), which in principle is excellently suited for compound annotation, suffers from limited sensitivity....
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...Mass spectrometry (MS)-based analysis of metabolites has become more and more advanced in recent years (Dettmer et al., 2007; Dunn, 2008; Griffiths and Wang, 2009; Allwood and Goodacre, 2010; Saito and Matsuda, 2010)....
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...…study was on the analysis and annotation of metabolites and lipids using high-resolution Fourier transform mass spectrometry (FT-MS), a technique that until recently has been restricted to specialized labs because of the high costs and technical complexity of the instrument (Dettmer et al., 2007)....
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TL;DR: In this paper, the methyl-tert-butyl ether (MTBE) extraction method was used for shotgun profiling of lipid species, and the results showed that the MTBE protocol delivered similar or better recoveries of species of most major lipid classes compared with the "gold-standard" Folch or Bligh and Dyer recipes.
1,632 citations