Tai Lieu Chat Luong Edited by Wolfram Weckwerth and Günter Kahl The Handbook of Plant Metabolomics Titles of the Series “Molecular Plant Biology Handbook Series” Kahl, G., Meksem, K (eds.) The Handbook of Plant Functional Genomics Concepts and Protocols 2008 ISBN: 978-3-527-31885-8 Meksem, K., Kahl, G (eds.) The Handbook of Plant Mutation Screening Mining of Natural and Induced Alleles 2010 ISBN: 978-3-527-32604-4 Meksem, K., Kahl, G (eds.) The Handbook of Plant Genome Mapping Genetic and Physical Mapping 2005 ISBN: 978-3-527-31116-3 Related Titles Harbers, M., Kahl, G (eds.) Tag-based Next Generation Sequencing 2012 ISBN: 978-3-527-32819-2 Hirt, H (ed.) Plant Stress Biology From Genomics to Systems Biology 2010 ISBN: 978-3-527-32290-9 Hayat, S., Mori, M., Pichtel, J., Ahmad, A (eds.) Nitric Oxide in Plant Physiology 2010 ISBN: 978-3-527-32519-1 Kahl, G The Dictionary of Genomics, Transcriptomics and Proteomics 2009 ISBN: 978-3-527-32073-8 Edited by Wolfram Weckwerth and Günter Kahl The Handbook of Plant Metabolomics The Editors Prof Dr Wolfram Weckwerth Universität Wien Molekulare Systembiologie Althanstr 14 1090 Wien Austria Prof Dr Günter Kahl Mohrmühlgasse 63500 Seligenstadt Germany Cover Legend The cover picture presents some structures of representative phytochemicals and biosynthetic pathways and enzymes of Arabidopsis thaliana, referred to in the chapter “Integrative analysis of secondary metabolism and transcript regulation in Arabidopsis thaliana” by Fumio Matsuda and Kazuki Saito (for further details see Chapter 9, Fig 4) The figure was originally published in “Matsuda, F., et al (2010) AtMeteEpress development: A phytochemical atlas of Arabidopsis development Plant Physiol, 152, 566–578), www.plantphysiol.org, # American Society of Plant Biologists The permission of the authors to partly use their figure in a changed format is greatly appreciated Foto of Arabidopsis: # Vasiliy Koval, Fotolia.com Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose No warranty can be created or extended by sales representatives or written sales materials The Advice and strategies contained herein may not be suitable for your situation You should consult with a professional where appropriate Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages Library of Congress Card No.: applied for British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at #2013 Wiley-VCH Verlag GmbH & Co KGaA, Boschstr 12, 69469 Weinheim, Germany Wiley-Blackwell is an imprint of John Wiley & Sons, formed by the merger of Wiley’s global Scientific, Technical, and Medical business with Blackwell Publishing All rights reserved (including those of translation into other languages) No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers Registered names, trademarks, etc used in this book, even when not specifically marked as such, are not to be considered unprotected by law Print ISBN: ePDF ISBN: ePub ISBN: mobi ISBN: oBook ISBN: 978-3-527-32777-5 978-3-527-66989-9 978-3-527-66990-5 978-3-527-66991-2 978-3-527-66988-2 Cover Design Adam-Design, Weinheim Typesetting Thomson Digital, Noida, India Printing and Binding Markono Print Media Pte Ltd, Singapore Printed in Singapore Printed on acid-free paper Dedicated to Ulrich and Hannelore Weckwerth for their endless sympathy, patience and guidance jVII Contents Preface XVII List of Contributors XIX Part I Central Metabolism 1 Metabolic Profiling of Plants by GC–MS Camilla B Hill and Ute Roessner Introduction Methods and Protocols Sample Preparation Sampling Homogenization and Extraction Procedure for Polar Extraction of Metabolites Chemical Derivatization: Methoxymation and Silylation Procedure for the Chemical Derivatization of Plant Extracts GC–MS Analysis 10 Procedure to Acquire GC–MS Data 11 Data Preprocessing and Export 12 Procedure for Postacquisition Data Preprocessing 12 Data Analysis and Statistics 14 Procedure for Postacquisition Data Analysis 15 Applications of the Technology 15 Perspectives 17 References 18 1.1 1.2 1.2.1 1.2.1.1 1.2.1.2 1.2.1.3 1.2.2 1.2.2.1 1.2.3 1.2.3.1 1.2.4 1.2.4.1 1.2.4.2 1.2.4.3 1.3 1.4 2.1 2.2 2.2.1 2.2.2 Isotopologue Profiling – Toward a Better Understanding of Metabolic Pathways 25 Wolfgang Eisenreich, Claudia Huber, Erika Kutzner, Nihat Knispel, and Nicholas Schramek Introduction 25 Methods and Protocols to Determine Isotopologues 31 Mass Spectrometry 31 Protocols for Isotopologue Profiling by GC–MS 36 VIII j Contents 2.2.2.1 2.2.2.2 2.2.2.3 2.2.3 2.2.4 2.2.5 2.2.6 2.3 2.3.1 2.3.2 2.4 Protein-Bound Amino Acids 36 Metabolic Intermediates and Polar Products 37 Carbohydrates 37 NMR Spectroscopy 38 Protocols for Isotopologue Profiling by NMR 41 Deconvolution of Isotopologue Data 43 Expanding the Metabolic Space by Retrobiosynthetic Analysis 45 Applications 46 Experiments Using ½U-13 C6 Glucose 46 Experiments Using 13 CO2 47 Perspectives 53 References 54 Nuclear Magnetic Resonance Spectroscopy for Plant Metabolite Profiling 57 Sonia van der Sar, Hye Kyong Kim, Axel Meissner, Robert Verpoorte, and Young Hae Choi Introduction 57 Methods and Protocols 59 Sample Preparation 59 Harvesting Plant Material 60 Drying 60 Extraction 60 Data Acquisition 60 Standard 1H-NMR Spectroscopy 61 J-Resolved Spectroscopy 61 Data Analysis 61 Applications 62 1D 1H-NMR Spectroscopy 62 2D NMR Spectroscopy 63 J-Resolved Spectroscopy 65 COSY and TOCSY 67 HMBC and HMQC/HSQC 68 NOESY or ROESY (CAMELSPIN) 69 DOSY 69 Magic Angle Spinning 70 Perspectives 71 References 72 3.1 3.2 3.2.1 3.2.1.1 3.2.1.2 3.2.1.3 3.2.2 3.2.3 3.2.4 3.2.5 3.3 3.3.1 3.3.2 3.3.2.1 3.3.2.2 3.3.2.3 3.3.2.4 3.3.2.5 3.3.3 3.4 4.1 4.2 Comprehensive Two-Dimensional Gas Chromatography for Metabolomics 77 Katja Dettmer, Martin F Almstetter, Christian J Wachsmuth, and Peter J Oefner Introduction 77 Methods and Protocols 81 Contents 4.2.1 4.2.2 4.2.3 4.2.4 4.2.5 4.3 4.3.1 4.3.2 4.4 Instrumentation 81 Sample Preparation and Analysis 82 Data Processing 83 Metabolic Fingerprinting 83 Quantitative Analysis of Selected Metabolites 84 Applications of the Technology 85 Data Analysis 85 Literature 88 Perspectives 89 References 90 MALDI Mass Spectrometric Imaging of Plants 93 Ale9s Svato9s and Hans-Peter Mock Introduction 93 Sample Preparation 96 Data Acquisition 98 Data Processing 98 Methods and Protocols 99 Sample Preparation and Handling 99 Intact Tissues 99 Cryosectioning 99 Matrix Deposition 100 Paintbrush (Figure 5.2) 100 Sublimation (Figure 5.3) 102 MALDI-MS Imaging Measurement 103 Bruker Ultraflex Instruments 103 Waters MALDI Micro MX 104 Imaging Intact Tissues and Objects 105 Future Perspectives 109 References 109 5.1 5.1.1 5.1.2 5.1.3 5.2 5.2.1 5.2.1.1 5.2.1.2 5.2.2 5.2.2.1 5.2.2.2 5.2.3 5.2.3.1 5.2.3.2 5.3 5.4 6.1 6.2 6.2.1 6.2.2 6.2.3 6.2.4 6.2.5 6.2.6 6.2.7 Medicago truncatula Root and Shoot Metabolomics: Protocol for the Investigation of the Primary Carbon and Nitrogen Metabolism Based on GC–MS 111 Vlora Mehmeti, Lena Fragner, and Stefanie Wienkoop Introduction 111 Methods and Protocols 112 Equipment and Software 112 Buffers and Chemicals 112 Plant Material and Harvest 113 Extraction 114 Derivatization 115 GC–MS Setup for the Analysis 115 Metabolite Identification and Quantification: Data Matrix Processing 116 jIX j Contents X 6.2.8 6.3 6.4 Data Mining 119 Applications of the Technology 119 Perspectives 121 References 123 Part II Secondary and Lipid Metabolism 125 Study of the Volatile Metabolome in Plant–Insect Interactions 127 Georg J.F Weingart, Nora C Lawo, Astrid Forneck, Rudolf Krska, and Rainer Schuhmacher Introduction 127 Plant–Insect Interactions 127 Significance of Volatile Plant Metabolites 128 Study of the Plant Volatile Metabolome in Plant–Insect Interactions 128 Setting Up of Biological Experiments 129 Sampling, Quenching, and Sample Preparation 130 Headspace Extraction and Measurement by GC–MS 131 Data Handling 134 Biological Interpretation 135 Methods and Protocols 135 Permanent Breed of Insects 135 Cultivation of Grapevine Plants and Inoculation with Phylloxera 136 Materials 136 Procedures 136 Sampling and Quenching of Plant Tissue (Roots and Leaves) 138 Sampling and Quenching of Root Tips 138 Sampling and Quenching of Grapevine Leaves 139 Milling and Weighing of Plant Tissue (Roots and Leaves) 140 Milling and Weighing of Root Samples 140 Milling and Weighing of Leaf Samples 141 Measurement – Automated HS-SPME Extraction and GC–MS Analysis 143 Materials 143 SPME Method 143 GC Method 144 MS Settings 144 Data Processing with AMDIS 145 An In-House Reference Library Has to be Established in Advance 145 Generation of RI Calibration File 146 Batch Job Analysis for the Simultaneous Processing of Multiple Sample Chromatograms 146 Statistics/Chemometrics 147 Univariate Statistics 147 Multivariate Statistics 148 7.1 7.1.1 7.1.2 7.1.3 7.1.3.1 7.1.3.2 7.1.3.3 7.1.3.4 7.1.3.5 7.2 7.2.1 7.2.2 7.2.2.1 7.2.2.2 7.2.3 7.2.3.1 7.2.3.2 7.2.4 7.2.4.1 7.2.4.2 7.2.5 7.2.5.1 7.2.5.2 7.2.5.3 7.2.5.4 7.2.6 7.2.6.1 7.2.6.2 7.2.6.3 7.2.7 7.2.7.1 7.2.7.2 Contents 7.3 7.4 Applications of the Technology 148 Perspectives 149 References 150 Metabolomics in Herbal Medicine Research 155 Lie-Fen Shyur, Chiu-Ping Liu, and Shih-Chang Chien Introduction 155 Methods and Protocols 158 Materials 158 Reagents 158 Equipment 159 Procedures 160 Sample Handling for Medicinal Plants 160 Sample Preparation for LC–MS Analysis 160 LC–MS Analysis 161 HPLC–Photodiode Array (PDA) MS Setup and Analysis 161 GC–MS Analysis 162 Plant Extract Preparation for GC–MS Analysis 163 GC–MS Parameters and Analysis 164 LC–MS and GC–MS Data Analysis 165 LC–SPE–NMR Analysis 166 Sample Preparation and LC–SPE–NMR Analysis 167 HPLC–SPE–NMR Data Analysis 168 Applications 168 Perspectives 169 References 170 8.1 8.2 8.2.1 8.2.1.1 8.2.1.2 8.2.2 8.2.2.1 8.2.2.2 8.2.2.3 8.2.2.4 8.2.2.5 8.2.2.6 8.2.2.7 8.2.2.8 8.2.2.9 8.2.2.10 8.2.2.11 8.3 8.4 9.1 9.2 9.2.1 9.2.1.1 9.2.1.2 9.2.1.3 9.2.2 9.2.2.1 9.2.2.2 9.2.3 9.2.3.1 9.2.3.2 9.2.3.3 Integrative Analysis of Secondary Metabolism and Transcript Regulation in Arabidopsis thaliana 175 Fumio Matsuda and Kazuki Saito Introduction 175 Methods and Protocols 177 Metabolome Analysis of Plant Secondary Metabolites 177 Sample Preparation 177 Data Acquisition 178 Preparation of Metabolite Accumulation Data from the Raw Chromatogram Data 179 Preparation of Combined Data Matrix 180 Preparation of Gene Expression Data 180 Combination of Data Matrices 180 Data Mining 180 BL-SOM Analysis 180 Correlation Analysis 181 Principal Component Analysis and Application of Other Data Mining Techniques 183 jXI Glossary phospholipids Triton X-100 is usually added to extraction buffers in order to destroy membranes that may entrap macromolecules such as DNA or RNA, and to solubilize proteins or protein complexes Two-dimensional blue-native/sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D blue-native SDS-PAGE): A technique for the analysis of protein complexes in their native state Briefly, cells are first lysed, the resulting protein mixture is dialyzed (to remove substances that interfere with subsequent steps), and the protein complexes are loaded with Coomassie Brilliant Blue (binds unspecifically to all proteins and leads to their overall negative charge) and separated from each other according to their size under native conditions in the first dimension The separated complexes are then in-gel denatured with sodium dodecyl sulfate (SDS), which leads to the decomposition of the protein complexes, and the individual proteins are separated by SDS-PAGE in the second dimension Two-dimensional difference gel electrophoresis (2-D DIGE, fluorescence difference gel electrophoresis): A variant of the conventional two-dimensional gel electrophoresis that allows the simultaneous separation of proteins from two samples and the identification of proteins specific for only one sample Briefly, the proteins from, for example, two different tissues are differentially labeled with the fluorochromes cyanine-3 and cyanine-5, respectively These fluorescent dyes not influence the relative migration of proteins during electrophoresis Then the prelabeled protein samples are mixed in equal quantities and co-separated by standard two-dimensional SDS-PAGE The gel is imaged for the two fluorophores, the resulting perfectly overlapping image is analyzed, and spots of interest (e.g., proteins only present in one sample) are excised from the gel, in-gel digested with trypsin, and the tryptic fragments are analyzed by mass spectrometry Two-dimensional gel electrophoresis (2D-electrophoresis, O’Farrell gel electrophoresis): A method for separating proteins on the basis of two of their characteristics: isoelectric point (pI) and molecular weight Usually, the protein mixture is first subjected to electrofocusing in a polyacrylamide matrix containing an ampholinesstabilized pH gradient This serves to separate the various proteins according to their pI Then the first-dimension gel is polymerized onto a second-dimension SDSpolyacrylamide slab gel in which the proteins are separated according to their molecular mass (O’Farrell gel) Two-dimensional ultraviolet polyacrylamide gel electrophoresis (2D-UV-PAGE): A technique for the visualization of proteins separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) without the use of dyes, but based only on autofluorescence of the proteins The intrinsic fluorescence of the proteins is excited by, for example, two different UV laser sources (such as a Ti:sapphire laser at 280 nm and an Nd:YAG laser at 266 nm), and the emitted fluorescence light is monitored with a highly sensitive CCD camera (the excitation light and the ambient light are filtered out) This label-free detection of proteins is dependent on aromatic side chains of the protein (e.g., tryptophan or tyrosine) and is as sensitive as silver staining (and 10 times more sensitive than the Coomassie Brilliant Blue staining) j409 410 j Glossary About 99% of all human proteins of more than 10 kDa contain at least one fluorescent amino acid and can therefore be detected by native fluorescence U, V Ultracentrifugation: The process of sedimenting cells, subcellular particles, or molecules in extreme gravity fields (e.g., more than 500 000g) through density gradients Ultracentrifuge: An instrument that drives rotors of various designs (e.g., fixedangle rotor, swing-out rotor, vertical rotor) at up to 100 000 rpm (corresponding to gravity fields of more than 500 000g) These gravitational forces serve to separate cells, subcellular particles, or molecules on the basis of either their density or size Preparative ultracentrifuges are used to prepare cells, subcellular particles, or molecules for their subsequent analysis Analytical ultracentrifuges allow the visualization of sedimenting particles in the spinning rotor through quartz windows and by means of UV or Schlieren optics Ultrahigh-throughput screening (uHTS): A robust routine process which allows more than 105 assays per day to be performed in a working volume of 1–10 ml per assay, using high-density platforms (e.g., 1536-well assay plates) Unnatural amino acid: Any synthetic amino acid that does not or only rarely occurs in Nature In most cases, unnatural amino acids have novel physicochemical and biological properties For example, 5-hydroxy-L-tryptophan is a fluorescent amino acid that is used as an oxidatively activated protein crosslinker For incorporation of unnatural amino acids into a growing peptide chain, new aminoacyl-tRNA synthetases must be engineered For this purpose, a synthetic 45-nucleotide long ribozyme (catalytic RNA) can charge transfer RNAs with virtually any unnatural amino acid Unnatural hydrocarbon chaincarrying amino acids can also be integrated into novel synthetic peptide drugs, and the hydrocarbon chains linked chemically, forcing the peptide into an a-helical conformation This conformation increases the stability of the peptide (especially its resistance to peptidases or proteases), its ability to move through membranes, and its intracellular activity W White biotechnology: A laboratory slang term for the application of the methodological repertoire of biotechnology to microorganisms (predominantly bacteria) White biotechnology aims at the industrial application of technologies (also isolated enzymes, or complete microbes in closed reactor systems) for the production of special biochemicals or biofuels Glossary Wild type (standard type): a) The most frequently occurring phenotype (strain, organism) in natural breeding populations b) The genetic constitution of an organism at the onset of recombinant DNA experiments with its genome or its plasmid(s) Hence this “wild type” is an arbitrarily specified genotype used as a basis for comparison in genetic programs X,Y X-ray crystallography: A technique for the determination of the three-dimensional structure of atoms or molecules in a crystal using the diffraction patterns produced by X-ray scattering XYZ arm (XYZ robotic arm): A part of a laboratory robot that moves in three dimensions (X–Y–Z) and allows, for example, liquid (or media, bacteria, clones) to be taken up in one microtiter plate, transferred into the wells of another microtiter plate (where, e.g., a reaction takes place), and the resulting mixture dispensed into the wells of yet another microtiter plate XYZ robotic workstation: An instrument for high-throughput experimentation that usually consists of a stage (holding pipetting heads, which can automatically be exchanged during operation, pipette tips, reagent containers, temperaturecontrolled blocks, in which, e.g., reactions are performed under controlled temperature regimes, and filtration blocks with filtering capacity in a 96-well format, or higher), and one or more movable arms with, for example, pipetting heads The stage can move in the X direction, while the arm and the pipetting head can move in the Y and Z direction, respectively This robotic workstation can be programmed, runs automatically, and has the potential to transfer reagents to and from 96-well (or larger) microtiter plates, mix them, incubate them, and dispense them In some instruments, the heads can be exchanged, and the workstation can then be modified to spot proteins or cDNAs onto glass slides XYZ robotic workstations are timesaving and continuously working parts of genomics laboratories Z Zero-integrated field electrophoresis (ZIFE): A variant of field inversion gel electrophoresis (FIGE) that is capable of resolving very large DNA molecules, but is very slow compared with FIGE Zero-mode waveguide (ZMW, zero-waveguide detector): Any hole in a 100 nm metal film deposited on a silicon dioxide substrate that has a diameter of some tens of nanometers and accommodates single molecules (e.g., DNA polymerases) in j411 412 j Glossary nanovolumes, allows reactions (e.g., a DNA synthesis reaction) to be run and reaction products to be visualized Since light cannot propagate through the small waveguides, they are called zero-mode waveguides For example, single DNA polymerase molecules can be immobilized on the bottom surface of such ZMWs, and these enzymes generate detectable pulses, as fluorescently labeled bases associate with the enzyme prior to their addition to a nascent DNA polymerized on the template DNA strand Briefly, nucleotides, each labeled with a different fluorochrome, are added to each ZMW Before being attached to the newly synthesized strand, each nucleotide remains bound to the enzyme for tens of milliseconds During this time, the attached fluorophore emits fluorescent light whose color identifies the corresponding base Then, the DNA polymerase cleaves off the fluorophore and the dye diffuses out of the detection volume Following incorporation, the signal immediately returns to the baseline, and the incorporation of the next nucleotide ensues Since thousands of ZMWs are located side-by-side, and each well contains a separate enzyme, thousands of processes can be monitored simultaneously in real time with a specially designed confocal imaging instrument The system forms the basis for one of the next-generation sequencing technologies [single-molecule real-time (SMRTTM) DNA sequencing technology; see http://www pacificbiosciences.com] Zinc cluster protein: A member of a class of nuclear proteins containing two zinc atoms that form a zinc cluster with six cysteine residues This specific cluster domain interacts with DNA (e.g., in the transcription factor GAL of Saccharomyces cerevisiae, which regulates the expression of genes encoding galactose-metabolizing enzymes) Zinc finger-associated domain (ZAD): A 90-amino acid specific domain of an insect-specific subfamily of the C2H2 zinc finger proteins that is located at the Nterminus and characterized by four invariant cysteine residues and four conserved sequence blocks linked by three variable spacer regions The domain is stabilized by the coordination of a Zn ion by the four cysteine residues In Drosophila melanogaster, about 100 zinc finger proteins each contain a ZAD These proteins probably evolved by a recent expansion of a ZAD-containing precursor gene, since the number of orthologous ZADs shared by D melanogaster and D pseudoobscura is 85, whereas D melanogaster and Anopheles gambiae have only six orthologous ZADs in common Zinc-finger nuclease (ZFN): A chimeric protein consisting of a synthetic DNArecognition domain with three C2H2 zinc fingers, fused to a nonspecific restriction endonuclease (usually FokI), that is used to generate double-strand breaks (DSBs) at specific genomic sites The introduction of such DSBs results in site-specific mutagenesis For example, transgenic Arabidopsis thaliana plants are engineered to contain a gene encoding a synthetic ZFN under the control of a heat-shock promoter, in addition to its associated target sequences Then heat-treatment is used to induce ZNF gene transcription The resulting ZFN proteins introduce DSBs, which are repaired by the plant’s nonhomologous end-joining (NHEJ) pathway, and Glossary in many cases lead to simple deletions and insertions at the repair site ZFNs are employed for targeted mutagenesis (gene targeting) in plants Zinc finger protein (ZFP): A member of a class of nuclear proteins containing from two to nine imperfect tandem repeats of the 30 amino acid sequence (Phe, Tyr)–Xaa–Cys–(Xaa)2(4)Cys–(Xaa)3–Phe–(Xaa)5–Leu–(Xaa)2–His–(Xaa)3–His–(Xaa)5, where Xaa represents any amino acid This region may fold into an independent structural domain organized by a tetrahedrally coordinated Zn2ỵ ion which binds the two Cys and the two His residues (“zinc finger”: His–His–loop, Cys–Cys–loop) Zinc fingers interact with DNA in such a way that the a-helix of each domain lies in the major groove, where sequence-specific contacts are made through residues of the finger motif (Lys13, Arg18, Arg21, and Lys24) and the polar side chains on the surface of the helix (Glu12, Ser14, Ser17, and Gln20) Alternatively to the Cys–Cys–loop or His–His–loop type of zinc fingers (e.g., TF III A-type), the Zn2ỵ ion may also be bound by four Cys ligands (in, e.g., steroid receptors and yeast transcription factors) Close to 700 human genes encode zinc finger proteins, and the Arabidopsis thaliana genome harbors 85 such genes Zinc finger protein binding region (ZIP): A sequence element of promoters (e.g., the human interleukin promoter) that serves as binding site for zinc finger proteins functioning as transcription factors (e.g., SP1) j413 j415 Index a b abiotic stress 16, 156, 158 acetyl-coenzyme A (CoA) 44 acidic sugars 199 Agilent Triple-Axis QUAD detector 11 aglycones 199, 200 alanine 38, 39, 62, 319 alfalfa 227 algae 215, 217 – lipid research, 223 alkaloid hermidin biosynthesis 49 alkamides 156, 161, 162 alkylhydroxylamine reagents AMDIS software 12, 13, 116, 134, 145, 146, 147, 330, 332 b-amyrin 24-hydroxylase (CYP93E1) 227 b-amyrin 30-hydroxylase (CYP72A154) 227 analysis of variance (ANOVA) 14, 89, 166, 262, 307, 308, 311, 313–315 antioxidants 197, 198 APCI See atmospheric pressure chemical ionization (APCI) Arabidopsis heat shock protein (HSP) 18.2 terminator 233 Arabidopsis thaliana 255 leaves – – mass spectrometric imaging 105, 106 – – optical image 107 – phytochemicals 184 arginine 290 artemisinin structures 47 AtMetExpress datasets 176 atmospheric pressure chemical ionization (APCI) 161, 202, 203, 291 Automated Mass Spectral Deconvolution and Identification System (AMDIS) 12, 13, 116, 134, 145–147, 325, 330, 332 batch-learning self-organizing map 177 bioactive phytocompounds 158 biodiesel 215, 217 biofuel 215 bioinformatics 5, 15, 95, 157, 207, 211 biological experiment setup 137 biological material 135 – Grapevine leaves, sampling and quenching 139, 140 – – materials 139 – – procedures 139, 140 – grapevine plants and inoculation cultivation with Phylloxera 136–138 – – infested root tips 139 – – inoculation with insects 137, 138 – – materials 136, 138 – – plants setup 137 – – plant tissue, sampling and quenching 138 – – procedures 136, 137, 138, 139 – – sampling and quenching of root tips 138, 139 – – uninfested root tips 139 – leaf samples, milling and weighing 141–143 – – materials 141 – – procedures 141–143 – measurement, automated HS-SPME extraction and GC–MS analysis 143–145 – – batch job analysis 146, 147 – – data processing with AMDIS 145, 146 – – GC method 144 – – in-house reference library 145, 146 – – materials 143 – – MS settings 144, 145 – – multivariate statistics 148 – – RI calibration file generation 146 – – SPME method 143 The Handbook of Plant Metabolomics, First Edition Edited by Wolfram Weckwerth and Günter Kahl
2013 Wiley-VCH Verlag GmbH & Co KGaA Published 2013 by Wiley-VCH Verlag GmbH & Co KGaA j Index 416 – – statistics/chemometrics 147 – – univariate statistics 147, 148 – for metabolomic analysis 199, 200 – permanent breed of insects 135 – perspectives 149, 150 – plant tissue, milling and weighing 140 – root samples, milling and weighing 140, 141 – – materials 140 – – procedures 140, 141 – technology applications 148, 149 Bonferroni–Holm adjustment 281 boron trifluoride (BF3) 324 Box–Cox algorithm 310 tert-butyldimethylsilyl-derivatized amino acids (TBDMS-amino) 31 c caffeic acid 161, 199 caffeoylquinic acids 161 Calvin cycle 44 CAMERA analysis 291 CaMV35S promoter 229 capillary electrophoresis (CE)–MS 17, 199 carbon isotope distribution 30 centaurein 161 chemical communication 93, 107 chemical derivatization – methoxymation and silylation – of plant extracts, procedure 9, 10 chemical fingerprinting 158 chlorogenic acid 161 ChromaTOF software 86 chromatographic fingerprint 169 chromatographic separation techniques cichoric acid 161 citrulline, spectrum and structure 290 13 C-labeled internal standards (ISs) 84 13 C-labeled metabolic intermediates 48 cloning vectors 230 13 C-NMR spectroscopy 68 13 CO2 experiments 50, 51 collision-induced dissociation (CID) MS/MS 95, 203 Comma Separated Values (CSVs) format 278 complex biological specimens See also biological material – qualitative and quantitative analyses 77 complex biosynthetic pathways 45 compound libraries 294 – exact mass search 291 comprehensive two-dimensional gas chromatography – applications of technology 85–90 – – data analysis 85–88 – – literature 88, 89 – – perspectives 89, 90 – data processing 83 – metabolic fingerprinting 83, 84 – for metabolomics 77–90 – methods and protocols 81–85 – – first-dimension column 81 – – instrumentation 81 – – second-dimension column 81 – sample preparation and analysis 82, 83 – selected metabolites, quantitative analysis 84, 85 – TOF-MS parameters 82 continuous chromatogram 79 conventional bench-top GC–MS instruments 89 correlation spectroscopy (COSY) 42, 67, 68, 168 COVAIN software 308 – adjusting outliers 310 – applications 314–320 – data preprocessing 308 – imputation of missing values 308, 310 – perspective 320 – scaling methods 310 Crassocephalum rubens 161 CryoProbes 40, 41, 58, 72 Cryosectioning 97, 99 Cytoscape 181, 320 d data acquisition 4, 11, 58, 60, 61, 79, 98, 104, 157, 177–179 data analysis 58, 61, 85, 112, 121, 134, 147, 169, 216, 305, 308, 310 – and statistics 14, 15 data matrix assembling 118 data preprocessing 83, 307–309, 313, 314 – commercial and free software packages 13 – and export 12–15 – on statistical analysis results, influences of 313 – – ANOVA 313 – – clustering 313 – – correlation coefficient 313 – – covariance 314 – – granger analysis 313 – – variance 314 2D DOSY 1H-NMR technique 70 decision trees (DTs) 322 deoxyhexoses 199 derivatization – chemical derivatization of plant extracts – chemical derivatization reactions 10 Index – methods in GC–MS FAME analysis 218 – products 116 – reagents 10 – standards 82 desorption electrospray ionization (DESI) 95 1D1H-NMR spectroscopy 62, 63 – disadvantage 62 diffusion ordered spectroscopy (DOSY) 65, 69, 70 dilution principle 48 direct analysis in real time (DART) 203 disease resistance 248 DNA separation 131 DNA sequences 15 2D NMR spectroscopy 58, 63–70, 65 – COSY and TOCSY 67, 68 – diffusion ordered spectroscopy (DOSY) 69, 70 – HMBC and HMQC/HSQC 68, 69 – J-resolved spectroscopy 65–67 – magic angle spinning 70, 71 – NOESY/ROESY (CAMELSPIN) 69 – perspectives 71, 72 drought stress experiment 119 drought stress-responsive shoot correlation 122 dry weight (DW) 119 dual-stage four-jet modulator 79 dual terminator (DT) fragment 233 dynamic headspace techniques 132 e Echinacea angustifolia 169 Echinacea pallida 169 Echinacea purpurea 169 electron-capture detectors (ECDs) 79 electron impact ionization (EI) 4, 11, 31, 33, 116, 144, 163, 165, 223, 321 electron ionization (EI) fragmentation 115 electrospray ionization (ESI) 33, 162, 178, 202, 203, 210, 291 enzyme-catalyzed reactions 30 Ephedra 169 estrogen 198 f FAME analysis 215 – applications 223 – bound and free fatty acids 217 – contaminants 219 – derivatization 219, 220 – extraction 216, 217 – GC–MS system 220 – – FAME reference mixture 224 – – instrument and conditions 223 – identification 220 – perspectives 223 – pigments 217–219 – protocols 216, 221, 222 – quantification procedure – – using C17:0 as an internal standard 222 – strategy by GC–MS 216 – – workflow 216 fast metabolite quantification (FMQ) 68 fatty acid methyl esters 215 ferulic acid 199 fingerprinting 57 flame-ionization detectors (FIDs) 79 flavonoid glyconjugates 199 flavonoid glycosides 161, 199 flavonoids 197 – biological material, preparation of – – for metabolomic analysis 199–201 – instrumental considerations 201–206 – isomeric 201 – metabolite profiling 199–201 – role and their derivatives in biological systems 197–199 – structures 198 Flex Imaging software v2.1 103 fossil fuels 215 Fourier transformation 61 Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer 4, 292 freeze-drying 131 fumarate calibration curves 87 g galactolipids 161 gas analyzer 52 gas chromatography (GC) 4, 31, 77, 78, 129, 215 gas chromatography–electron ionization mass spectrometry (GC–EI-MS) 158 gas chromatography-mass spectrometry (GCMS) 4, 6, 10–12, 62, 111, 131, 143, 155, 156 – data, procedure 11, 12 – experimental steps – methanol extraction 164 – supercritical fluid extraction (SFE) 163 GC·GC data visualization 80 GC–GC setup scheme 78 GC·GC–TOF-MS chromatograms 85 GC–MS-based isotopologue profiling 31, 35 GC–MS-based metabolomics 15, 17 GC–TOF-MS instruments 11 Gene Expression Data 180 genes encoding sulfotransferases 175 j417 j Index 418 genetic alterations 16 genetic genomics 17 genetic linkage 255 genetic markers 157 genistein 198 genome sequencing 57 genome-wide association (GWA) approach 255 – applications 267, 268 – biological question to be addressed 256 – candidate – – gene selection 265, 266 – – gene validation 266, 267 – chemical class 256, 257 – chemistry to study 256 – computational platform to use for analysis 261–265 – conducting – – appropriate follow-up experiments 260 – – phenotype measurements 261 – domestication status 260 – extraction and detection platform 257, 258 – genotypic choices 258 – methods 256 – perspectives 268 – populations available 259, 260 – protocols 256 – species choices 258 ginkgolide structures 46, 47 ginseng products 53, 169 glucose – isotopologue composition 44 – stable carbon isotopologues 26–28 glucosinolates 105, 175 glucuronic acid 199 Glycine max See soybean glycyrrhetinic acid 228, 229 Glycyrrhiza uralensis 227 glycyrrhizin 227, 228 GMD See Golm metabolome database GO categories 181 Golm metabolome database 321, 322 – applications 341, 342 – concept of chemical compound 323 – data entities 322, 324, 325 – decision tree (DT)-supported substructure prediction 329 – interpreting mass spectral analysis results 329–336 – mass spectral matching 326–329 – mass spectrum query submission and analysis options 325, 326 – perspectives 341, 342 – roles of compounds 323 – text search queries 325 – web services 336–341 greenhouse experiments 129 h hampers data analysis 86 headspace analysis 131 headspace extraction techniques 132 headspace (HS)-SPME 132 heavy metal 158 herbicide 158 hermidin biosynthesis 52 heteronuclear multiple bond correlation 68 heteronuclear multiple quantum coherence (HMQC) 42, 65, 68 heteronuclear single quantum coherence (HSQC) 42, 68, 69, 168 hexoses 199 hierarchical cluster analysis (HCA) 14 high chromatographic resolution 77 high-performance liquid chromatography (HPLC) 4, 178 high-performance liquid chromatography (HPLC) coupled with NMR (HPLC–NMR) 157 high-performance liquid chromatography (HPLC)–MS 33, 199 high-resolution 13C NMR spectroscopy 38 high-resolution magic angle spinning (HR-MAS) NMR spectroscopy 70 high-resolution spectra 70 high-throughput profiling 89 high-throughput tissue homogenizer H NMR-based metabolite profiling 16 H-NMR chemical shifts 63 H-NMR spectroscopy 63, 68, 70 – applications 64 holistic metabolic fingerprinting 80 homogenization 7, 8, 140, 142 homonuclear Hartmann–Hahn (HOHAHA) experiment 68 HPLC–SPE–NMR data analysis 168 HR-MAS NMR spectroscopy 71 HS-SPME GC–MS-based metabolomics methods 149 HumanMetabolome Database (HMDB) 300 30-hydroxy-11-oxo-b-amyrin 229 hyperforin – formation 49 – structures 47 i ICA See independent component analysis (ICA) imaging data processing software 97 Index INADEQUATE experiments 43 independent component analysis (ICA) 119, 307, 308, 312, 314 indium tin oxide (ITO)-coated glass slides 99 induced defense 127 in silico library search 294 integrated analysis workflow, of metabolome and transcriptome data 176 isoflavones 198 isopentenyl diphosphate (IPP)/dimethylallyl diphosphate (DMAPP) precursors 43 “isoprene rule” 45 isopropylidene/acetate derivative 53 isotopologue profiling 25 – applications 46–54 – carbohydrates 37 – determine isotopologues, methods and protocols 31–46 – feature 30 – isotopologue data deconvolution 43–45 – mass spectrometry 31–35 – metabolic intermediates 34, 37 – metabolic space expanding by retrobiosynthetic analysis 45, 46 – monosaccharides 35 – NMR spectroscopy 38–41 – perspectives 54 – plants 47 – polar products 37 – protein-bound amino acids 36 – protocols – – by GC-MS 36, 37 – – by NMR 41–43 – stable carbon isotopologues of glucose 26–28 – TBDMS-amino acids 32 isotopomers 29 – quantification 32 j jacein 161 J-resolved technique 61, 65, 67 k kaempferol 3-O-rhamnosyl-(1!6)galactoside 161 K-prototype profiles 276 l laser ablation electrospray ionization (LAESI) 96 laser microdissection (LMD) methods 96 LC–SPE–NMR methods 158 – sample preparation 167, 168 LDI imaging method 105 LECO ChromaTOF software 83 licorice 227 licorice b-amyrin 11-hydroxylase (CYP88D6) 227 limit of detection (LOD) 84 linkage disequilibrium (LD) 255 lipoxygenase (LOX) pathway 148 liquid chromatography (LC) 4, 159, 201, 211, 248 liquid chromatography–electrospray ionization mass spectrometry (LC–ESI-MS) 158 liquid chromatography-mass spectrometry (LC-MS) 4, 62, 111, 149, 155, 161 – autosampler 160 – based shotgun proteomics 89 – data file 98 – metabolome analysis with 156, 157 liquid extraction surface analysis (LESA) 96 liquid nitrogen 130 lithium dihydroxybenzoate matrix 101 lower limit of quantification (LLOQ) 86 low-temperature probes 58 m magic angle spinning 70, 71 magnetic resonance imaging (MRI) 71 MALDI imaging experiments 98, 101, 203 MALDI mass spectrometric imaging of plants – Bruker ultraflex instruments 103–104 – cryosectioning 99–100 – data acquisition 97, 98 – data processing 98 – ImagePrep 101–102 – imaging intact tissues and objects 105–108 – MALDI-MS imaging measurement 103, 104 – matrix deposition 100–103 – methods and protocols 99–104 – negatively charged molecules, matrix application 100, 101 – neutral lipids, matrix application 101 – paintbrush 100 – perspectives 109 – sample preparation 96, 97 – – and handling 99, 100 – sublimation 102–103 – Waters MALDI Micro MX 104 MALDI matrix 96, 97, 105 MALDI micro MX mass spectrometer 104 MALDI-MS imaging 98, 108 MALDI stainless-steel target plate 100 MALDI-TOF mass spectra 98, 101, 106, 107 malonic acid 199 marker-assisted selection (MAS) 248 j419 j Index 420 MarkerLynx 281 MarVis tool 277 MassBank 300–302 mass spectral tags (MSTs) 321 – databases 249 mass spectrometry (MS) 3, 4, 29, 77, 93, 105, 111, 129, 215 mass spectrometry imaging (MSI) methods 93–95, 97, 98, 100, 103, 107, 108 – principles 94 mass-to-charge ratio (m/z) 111, 275 MATLAB 84, 112, 118, 314 Matrix Analyzer 14 Medicago sativa 227 Medicago truncatula root and shoot metabolomics 111–122 – buffers and chemicals 112, 113 – data mining 119 – derivatization 115 – equipment and software 112 – extraction 114, 115 – GC–MS setup for analysis 115, 116, 120 – metabolite identification and quantification, data matrix processing 116–118 – methods and protocols 112–119 – perspectives 121, 122 – plant material and harvest 113, 114 – technology, applications 119–121 medicinal properties, of herbs 168 MetaboAnalyst 157 metabolic cycle 131 metabolic fingerprinting 83, 84 – data processing flowchart 85 metabolic fluxes 25 – determination 25 metabolic network 30 – comprehensive analysis 54 metabolic profiling – methods and protocols 7–15 – perspectives 17, 18 – plants by GC–MS 3–18 – sample preparation 7–9 – – chemical derivatization, methoxymation and silylation – – chemical derivatization of plant extracts, procedure 9, 10 – – data analysis and statistics 14, 15 – – data preprocessing and export 12–15 – – GC–MS analysis 10–12 – – homogenization and extraction 7, – – polar extraction of metabolites, procedure 8, – – postacquisition data analysis procedure 15 – – postacquisition data preprocessing procedure 12–14 – – sampling – technology applications 15–17 metabolites – annotation and identification of 290, 291 – annotation tool 177 – based clustering 275 – CAMERA analysis 291 – elemental composition from MS 292–294 – levels of identification, with levels of confidence 289, 290 – profiling 199–201 – – advantages 249 – reference spectral library 299–302 – in silico library search 294–299 – structural classes metabolome analysis, of plant secondary metabolites 177 – application of other data mining techniques 183 – batchlearning self-organizing map, data visualization by 183, 184 – BL-SOM analysis 180 – correlation analysis 181, 182 – data acquisition 178, 179 – data mining 180 – integrated analysis of transcriptome 185 – – advantages 186, 187 – metabolome (AtMetExpress) data 185 – perspectives 187–190 – preparation of combined data matrix 180 – preparation of metabolite accumulation data 179, 180 – principal component analysis 183 – sample preparation 177 metabolome data – acquisition 177 – integrated analysis workflow 176 metabolomics 3, 57, 111, 155 – applications 168, 169 – common biological questions in 305–308 – data interpretation 157 – equipment 159, 160 – GC–MS parameters and analysis 162–165 – in herbal medicine research 169 – HPLC–photodiode array (PDA) MS 161, 162 – HPLC–SPE–NMR data analysis 168 – LC–MS and GC–MS data analysis 161, 165, 166 – LC–SPE–NMR analysis 166, 167 – methods and protocols, for metabolite profiling 158 – perspectives 169, 170 Index – plant extract preparation 163, 164 – project – reagents 158, 159 – sample handling for medicinal plants 160 – sample preparation – – for LC–MS analysis 160, 161 – – and LC–SPE–NMR analysis 167, 168 – workflow 129 metabolomics-assisted plant breeding 247–249 – applications 251–253 – methods 249–251 – perspective 253, 254 Metabolomics Standards Initiative (MSI) 289 metaP-Serve 157 MetFrag system 294–299 MetFrag Web application 296 methoxylamine hydrochloride 115 methoxymation N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (MTBSTFA) 9, 36 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway 148 N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) 9, 37, 82, 112 Metlin database 300 mevalonate (MEV) 148 – pathway 46, 47 MPS2XL GC–MS autosampler multidimensional gas chromatography (MD-GC) 78 MultiExperiment Viewer (MeV) 183 multiple 13C-labeled glucose isotopologues – 13C NMR signatures 40 multiple headspace extraction (MHE) 133 multivariate statistical methods 177, 312 – clustering 312, 313 – independent component analysis 312 – principal component analysis 312 MYB transcription factors 175 n National Institute of Standards and Technology (NIST) 12, 132 natural products 30, 44 Nd:YAG lasers 95 network Common Data Form (NetCDF) format 179 network-level analysis 313 – network inference 313 – network mapping 313 N-fertilized metabolites 121 N-fixing plants 119 nicotine – biosynthesis 50 – isotopologue profiles 47 NIST retention index 117, 118 N-methyl-N-bis(trifluoroacetamide) (MBTFA) 324 N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) 324 N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) 324 nocturnal rhythm 130 nonsupervised statistical method 148 nonvolatile metabolites 31 nopaline synthase terminator (NOS) 228 nuclear magnetic resonance (NMR) – based plant metabolomics 62 – – sample preparation protocols 59 – metabolomic analysis 65 – signal patterns 38, 41 – spectroscopy 29, 35, 40, 57–72, 58, 155, 156, 157, 289 – – development 72 nuclear Overhauser enhancement (NOE) 69 nuclear Overhauser enhancement spectroscopy (NOESY) 42, 69, 168 o oligosaccharides 163, 199 one-dimensional SOMs (1D-SOMs) 276 – active prototype/marker plot 284 – applications 281–286 – cluster analysis 280, 281 – clustering 277–280 – data import 277 – marker scatter plot 284 – perspectives 286 – representation of marker candidates 282–284 – visualization 276 – WT/DcsnE data set 285 optimal cutting temperature (OCT) compound 97 Orbitrap analyzer 95 organic acids 3–5, 58, 62, 68, 88, 111, 119, 121, 156, 251, 324 overlapping peak 77 2,3-oxidosqualene, 227 11-oxo-b-amyrin 233 p P450 genes 233 parallel factor analysis (PARAFAC) 88 partial least squares (PLS) 15 j421 j Index 422 partial least squares-discriminant analysis (PLS-DA) 58 partition chromatography PCA See principal component analysis (PCA) p-coumaric 199 Pearson correlation coefficients (PCCs) 181 PEG oligomers 103 pentasaccharides 199 pentoses 199 perfluorotributylamine (PFTBA) 144 perlite–vermiculite mixture 113 phenolics 161 phenylpropenoic acids 199 phosphodiesterase-5 (PDE-5) inhibitors 70 phosphoenolpyruvate (PEP) 45 photobioreactors 215 photodiode arrays (PDAs) 289 photosynthetic fixation products 48 photosynthetic photon flux density (PPFD) 48 phylloxera 135 phytomedicines 170 PhytomicsQC platform 169 phytopreparations 170 plant breeding 247 – natural variations, of species 248 – technology, for monitoring and selection for traits 248, 249 plant-derived extracts 17 plant–insect interactions See volatile metabolome plant metabolite analysis – sample preparation for – strategies for plant metabolite profiling 6, 10 – applications 62–71 – data acquisition 60, 61 – data analysis 61 – 1D1H-NMR spectroscopy 62, 63 – 2D NMR spectroscopy 63–70 – homogenization and polar extraction – J-resolved spectroscopy 61 – methods and protocols 59–61 – nuclear magnetic resonance spectroscopy for 57–72 – sample preparation 59, 60 – – drying 60 – – extraction 60 – – harvesting plant material 60 – standard 1H-NMR spectroscopy 61 plants – chemical profiling, sampling strategies 97 – chromatogram 14 – MALDI mass spectrometric imaging 93–108 – metabolic composition – metabolome analysis 71, 156 – – secondary metabolites 177, 178 – proteome 58 plasmid construction – in multi-gene transformation 233 – – application of technology, 242 243 – – perspectives 243 – – primers used for 234, 235 – pHSG299 CSPS 35S-CYP72-DT2 237 – pHSG299 CSPS 35S-CYP88-DT 236, 237 – pHSG299-CYP93(RNAi)-DT 238, 239 – pUHR KS CSPS Thsp 236 – pUHR KS CSPS Thsp-CYP88-CYP72-CYP93 (RNAi) 239 – three-key P450 genes, construction of 229 polar metabolites 8, 9, 149 polystyrene boxes 140, 141 portable 13CO2 incubation unit – schematic drawing 52 postacquisition data – analysis procedure 15 – preprocessing procedure 12–14 postgenomics technology 156 principal component analysis (PCA) 8, 14, 15, 58, 61, 88, 119, 134, 147, 148, 168, 276, 308, 311, 312, 315, 316, 320 programmed temperature vaporization (PTV) injector 81 proof-of-principle experiment 53 protein-bound amino acids 31 pulse–chase experiments 49 q QC samples 145 QSTAR QqTOF instrument 292 quadruplicate metabolome data 177 quality control (QC) samples 130 quantitative mass (QM) 117, 118 quantitative trait loci (QTLs) analysis 16 quercetin 3-O-rhamnosyl-(1!6)galactoside 161 r radiofrequency (RF) 11 RAMP energy mode 294 rapid oxidative reaction (ROS) 109 README.txt file 277 reference spectral library 299–302 regression analysis 87 relative isotope abundance (RIA) 292 relative standard deviation (RSD) 86 retention index (RIs) 5, 134, 321 – calibration file 146 Index retention time (RT) 5, 111, 117, 118, 275 retention time index (RI) systems, calculations 12 retention time locking (RTL) 11 retrobiosynthetic analysis, of metabolic intermediates 46 reverse-phase liquid chromatography (LC)–high-resolution MS 157 RIKEN Metabolomics web site 179 rotating frame Overhauser enhancement spectroscopy (ROESY) 69 rutin 161 s sample handling, for medicinal plants 160 sample matrix 80 sample preparation 7, 12, 58, 59, 60, 77, 80, 96–97, 128–131, 177, 249 – and analysis 82 – drying 60 – extraction 60 – and handling 99 – harvesting plant material 60 – for LC–MS analysis 160 – and LC–SPE–NMR analysis 167 – strategies 97 secondary ion mass spectrometry (SIMS) 95 selected ion chromatograms (SICs) 104 selective reaction monitoring (SRM) 115 self-organization mapping (SOM) 15, 276 signal-to-noise ratio (S/N) value 83, 311 silylation SIMCA-P software 61 sinapinic acid 199 single-cell imaging 96 single ion monitoring (SIM) 33 single nucleotide polymorphisms (SNP) technology 248 SIRIUS software 292, 294, 298 – advantages 295 – applications – – with isotope analysis of vitexin-2-Orhamnoside 293, 294 – fragmentation tree of citrulline 295 software packages, SIMCA-Pỵ and SAS 157 solid-phase microextraction (SPME) 132134, 143, 145, 149 – efficiency 133 – fiber coatings 133 solvents description 113 soyasapogenol B biosynthetis 228 soyasaponin I 227, 228 soybean 227 – conditions of particle bombardment 240 – transformation by particle bombardment 239, 240 – transgenic plants, selection and generation of 240, 241 specific quality parameters 157 SPME See solid-phase microextraction (SPME) stable isotope-labeled compounds Staedtler Triplus gel-liner 100 standard chromatogram 88 standard 1H-NMR spectroscopy 61 starch biosynthesis 46 static headspace extraction 132 stationary 13CO2 incubation chamber 49 stereo-microscope 100 stir-bar sorptive extraction (SBSE) 132, 134 stress tolerance 248 sublimation 102–103 sucrose isotopologue profiling 53 super-long-range COSY experiment 67 systematic acquired resistance (SAR) 67 systemic defense 127 t tandem mass spectra 158 target component library (TCL) 14 time-of-flight (TOF) mass spectrometers 79, 95, 115, 161 total correlation spectroscopy (TOCSY) 67 total ion current (TIC) chromatogram 81 traditional Chinese medicine (TCM) 168 – quality control 169 transcriptome data 180, 181, 187, 189 – integrated analysis workflow 176 triterpene aglycone b-amyrin 227 triterpene glycone – GC-MS analysis for 241, 242 – GC-MS conditions 242 triterpene saponin glycones 227, 228 tri-tert-butyldimethylsilyl (TBDMS) derivatives trifluoroacetic acid (TFA) 101 trimethylchlorosilane (TMCS) TriPlus autosampler 116 t-test 14 two-dimensional gas chromatography 77, 78 u ultra performance liquid chromatography (UPLC) 178, 281 ULTRA-TURRAX tissue homogenizers j423 j Index 424 uni- and bivariate statistical methods 311 – ANOVA 311 – correlation coefficients 311 – Granger causality analysis 311, 312 unscrambler statistical software (CAMO) 15 urea cycle 290 v vitamin E 256 vitexin-2-O-rhamnoside, spectrum and structure 291 volatile metabolome 135 – biological experiments, setting up 129, 130 – biological interpretation 135 – biological material 135 – data analysis 134 – data handling 134, 135 – data processing 134 – GC–MS, headspace extraction and measurement 131, 132 – methods and protocols 135–148 – perspectives, 149, 150 – plant material, sampling and quenching 130, 131 – sample preparation 131 – sampling, quenching, and sample preparation 130, 131 – significance 128 – solid-phase microextraction (SPME) 132–134 volatile organic compounds (VOCs) 128, 149 w web-based metabolomic data tools 15 x XCMS Online server 291