Metabolomics in Practice -- Contents -- List of Contributors -- Preface -- 1 The Sampling and Sample Preparation Problem in Microbial Metabolomics -- 1.1 Introduction -- 1.2 Microorganisms and Their Properties -- 1.3 Sampling Methods -- 1.3.1 The Need for Rapid Sampling -- 1.3.2 Sampling Systems -- 1.4 Quenching -- 1.4.1 Quenching Procedures and Their Properties -- 1.4.2 Validation of the Quenching Procedure and Minimization of Metabolite Leakage -- 1.4.3 Quenching Procedure for Determination of Intracellular Metabolites in the Presence of Extracellular Abundance -- 1.4.4 Quenching of Bacteria -- 1.5 Metabolite Extraction -- 1.5.1 Extraction Methods and Their Properties -- 1.5.2 Validation of Extraction Methods for Yeast Metabolomics -- 1.6 Application of 13C-Labeled Internal Standards -- 1.7 Conclusions -- References -- 2 Tandem Mass Spectrometry Hyphenated with HPLC and UHPLC for Targeted Metabolomics -- 2.1 Introduction -- 2.2 LC-MS-Based Targeted Metabolomics -- 2.3 Liquid Chromatography -- 2.4 Mass Spectrometry -- 2.4.1 Ionization Techniques -- 2.4.2 Mass Analyzers -- 2.5 Sample Preparation -- 2.6 Relative and Absolute Quantification -- 2.7 Applications -- 2.8 Synopsis -- References -- 3 Uncertainty of Measurement in Quantitative Metabolomics -- 3.1 Introduction -- 3.1.1 MS-Based Techniques in Metabolomics -- 3.1.2 Uncertainty of Measurement in Quantitative Analysis -- 3.1.2.1 Definition -- 3.1.2.2 Uncertainty Calculation According to the Bottom-Up Approach -- 3.2 Uncertainties of Quantitative MS Experiments -- 3.2.1 Uncertainties in Sample Preparation -- 3.2.1.1 Sampling and Sample Preparation in Metabolite Profiling in Fermentations -- 3.2.1.2 Calculation of Sample Preparation Uncertainty for Intracellular Metabolite Quantitation in Yeast: A Practical Example -- 3.2.1.3 LC-MS.

3.2.2 Uncertainty of Mass Spectrometric Assays (LC-MS and GC-MS Measurements) -- 3.2.2.1 GC-MS -- 3.2.2.2 Calculation of Uncertainty for LC-MS Measurements of Cell Extracts: A Practical Example -- 3.3 Concluding Remarks -- Abbreviations -- Acknowledgment -- References -- 4 Gas Chromatography and Comprehensive Two-Dimensional Gas Chromatography Hyphenated with Mass Spectrometry for Targeted and Nontargeted Metabolomics -- 4.1 Introduction and Scope -- 4.2 Sample Preparation for GC-Based Metabolite Profiling -- 4.3 GC-MS and GC x GC-TOFMS Instrumentation for Metabolomics -- 4.4 Data Analysis Strategies and Software -- 4.5 Illustrative Examples and Concluding Remarks -- References -- 5 LC-MS-Based Nontargeted Metabolomics -- 5.1 Introduction -- 5.2 LC-MS-Based Untargeted Metabolomics -- 5.2.1 LC Issues -- 5.2.2 Mass Spectrometry -- 5.3 Study Design -- 5.4 Sample Preparation -- 5.5 Analytical Strategies -- 5.6 Data Analysis -- 5.7 Metabolite Identification -- 5.8 Applications -- 5.9 Synopsis -- References -- 6 The Potential of Ultrahigh Resolution MS (FTICR-MS) in Metabolomics -- 6.1 Introduction -- 6.2 Metabolomics Technologies -- 6.3 Principles of FTICR-MS -- 6.3.1 Natural Ion Movement Inside an ICR Cell Subjected to Magnetic and Electric Fields -- 6.3.2 Applied Physical Techniques in FTICR-MS -- 6.3.3 Practical Advantages of FTICR-MS -- 6.4 Proceeding in Metabolomics -- 6.4.1 Network Analysis and NetCalc Composition Assignment -- 6.4.2 Statistics on FTICR-MS Datasets -- 6.5 Application Example in Metabolomics Using FTICR-MS Exhaled Breath Condensate -- 6.5.1 The Experiment -- 6.5.2 FT-ICR/MS Measurement -- 6.5.3 Data Preprocessing -- 6.5.4 C-H-N-O-S-P Formula Annotation -- 6.5.5 Statistical Analysis -- 6.5.5.1 Statistical Preprocessing -- 6.5.6 Synthesis of Biochemical Mass Difference Networking and Statistical Results -- 6.6 Conclusion and Remarks.

References -- 7 The Art and Practice of Lipidomics -- Abbreviations -- 7.1 Introduction -- 7.2 Lipid Diversity -- 7.3 Tackling the Lipidome: State-of-the-Art -- 7.4 LC-MS-Based Lipidomics -- 7.4.1 Lipid Extraction -- 7.4.1.1 Biological Fluids and Cellular Material -- 7.4.1.2 Skin (Stratum Corneum) -- 7.4.1.3 Solid-Phase Extraction (SPE) -- 7.4.2 LC-MS(/MS) -- 7.4.2.1 Retention Time Characteristics -- 7.4.2.2 Ionization Characteristics -- 7.4.2.3 Identification of Lipids -- 7.4.3 Data Processing and Analysis -- 7.5 GC-MS-Based Lipidomics -- 7.5.1 Sample Preparation -- 7.5.2 GC-MS -- 7.5.3 Data Processing and Analysis -- 7.6 Conclusion -- References -- 8 The Role of CE-MS in Metabolomics -- Abbreviations -- 8.1 Introduction -- 8.2 CE-MS -- 8.2.1 CE Separation Conditions -- 8.2.2 CE-MS Coupling -- 8.2.2.1 Interfacing -- 8.2.2.2 Mass Analyzers -- 8.3 Sample Pretreatment -- 8.4 Data Analysis -- 8.5 Applications -- 8.5.1 Targeted Approaches -- 8.5.2 Nontargeted Approaches -- 8.6 Conclusions and Perspectives -- References -- 9 NMR-Based Metabolomics Analysis -- 9.1 Introduction -- 9.2 Platforms for Metabolomics -- 9.2.1 Mass Spectrometry (MS) -- 9.2.1.1 Gas Chromatography-Mass Spectrometry (GC-MS) -- 9.2.1.2 Liquid Chromatography-Mass Spectrometry (LC-MS) -- 9.2.1.3 Capillary Electrophoresis-Mass Spectrometry (CE-MS) -- 9.2.1.4 Fourier Transform-Ion Cyclotron Resonance-Mass Spectrometry (FT-ICR-MS) -- 9.2.2 Fourier Transform-Infrared Spectroscopy (FT-IR) -- 9.2.3 Nuclear Magnetic Resonance Spectroscopy: Principles and Techniques -- 9.2.3.1 One-Dimensional Nuclear Magnetic Resonance (1H and 13C NMR) -- 9.2.3.2 J-Resolved Spectroscopy (JRES) -- 9.2.3.3 Correlation Spectroscopy (COSY) -- 9.2.3.4 Total Correlation Spectroscopy (TOCSY) -- 9.2.3.5 Heteronuclear Two-Dimensional Methods -- 9.2.3.6 Combined Two-Dimensional Methods -- 9.3 NMR for Metabolomics.

9.3.1 Sample Preparation -- 9.3.2 Metabolite Identification -- 9.3.3 Data Analysis: Turning Data into Information, Possibly Knowledge -- 9.3.3.1 Data Preprocessing -- 9.3.3.2 Principal Component Analysis (PCA) -- 9.3.3.3 Partial Least Squares (PLS) Projections to Latent Structures -- 9.3.3.4 Bidirectional Orthogonal-PLS (O2PLS) -- 9.3.3.5 Validation -- 9.4 Applications of NMR-Based Metabolomics -- 9.4.1 Understanding Stress Response -- 9.4.2 Application to Bioactivity Screening -- 9.4.3 Quality Control of Herbal Medicines -- 9.4.4 Chemotaxonomy -- 9.4.5 Agricultural Applications -- 9.5 Future Prospects and Conclusions -- References -- 10 Potential of Microfluidics and Single Cell Analysis in Metabolomics (Micrometabolomics) -- 10.1 Introduction -- 10.2 Sample Processing for Metabolomics -- 10.2.1 Solid Phase Extraction -- 10.2.2 Laminar Diffusion -- 10.2.3 Fluidic Pumping for On-Chip Mixing -- 10.3 Microfluidic Separations for Metabolic Analysis -- 10.3.1 Microchip Capillary Electrophoresis -- 10.3.1.1 MCE Systems -- 10.3.1.2 Sample Injection -- 10.3.1.3 Electrophoretic Separations -- 10.3.2 Analyte Detection -- 10.3.2.1 Optical Detection -- 10.3.2.2 Electrochemical Detection -- 10.4 Microfluidics for Cellular Analysis -- 10.4.1 Requirements for Single Cell Metabolomics -- 10.4.2 Types of Microfluidic Instrumentation -- 10.4.3 Biological Questions -- 10.4.3.1 Monitoring Metabolic Response to Stimulation and Cell-to-Cell Signaling -- 10.4.3.2 Pharmacokinetics/Pharmacodynamics -- 10.4.3.3 Clinical Diagnostics -- 10.5 A Look Forward -- References -- 11 Data Processing in Metabolomics -- 11.1 Introduction and Scope -- 11.2 Characteristics of Metabolomics Data -- 11.2.1 Correlation Structure of Metabolomics Data -- 11.2.2 Informative versus Noninformative Variation -- 11.2.3 Low Samples-to-Variables Ratio -- 11.2.4 Measurement Error -- 11.2.5 Dynamics.

11.2.6 Nonlinear Relations -- 11.3 Types of Biological Questions Asked -- 11.3.1 Methods Should Follow the Questions -- 11.3.2 Biomarkers -- 11.3.3 Treatment Effects -- 11.3.4 Networks and Mechanistic Insight -- 11.4 Validation -- 11.4.1 Several Levels of Validation -- 11.4.2 Curse of Dimensionality -- 11.4.3 Cross-Validation and Permutations -- 11.5 Overview of Methods -- 11.5.1 Exploratory Analysis -- 11.5.2 ANOVA and Other Univariate Methods -- 11.5.3 Advanced Exploratory Analysis -- 11.5.4 Regression Methods -- 11.5.5 Discriminant Analysis -- 11.5.6 Multilevel Approaches -- 11.5.7 Network Inference -- References -- 12 Metabolic Flux Analysis -- 12.1 Introduction -- 12.2 Prerequisites for Flux Studies -- 12.2.1 Network Topology and Cellular Composition -- 12.2.2 Network Formulation and Condensation -- 12.2.3 Metabolic and Isotopic Steady State -- 12.2.4 Definition of Isotope Labeling Patterns -- 12.3 Stoichiometric Flux Analysis -- 12.4 Labeling Studies Using Isotopes -- 12.4.1 Radiolabeled Isotopes -- 12.4.2 Stable Isotopes -- 12.5 State-of-Art 13C Flux Analysis -- 12.5.1 Modeling of Carbon Transitions -- 12.5.2 Experimental Design -- 12.5.3 Flux Calculation and Statistical Evaluation of Flux Data -- 12.5.4 Labeling Analysis by Mass Spectrometry -- 12.5.5 Labeling Analysis by Nuclear Magnetic Resonance Spectroscopy -- 12.6 Application of Metabolic Flux Analysis -- 12.6.1 Improvement of Industrial Production Strains -- 12.6.2 Integration into Systems Biology Approaches -- 12.7 Recent Advances in the Field -- 12.7.1 High-Throughput Flux Screening -- 12.7.2 Flux Dynamics -- 12.8 Concluding Remarks -- Acknowledgments -- References -- 13 Metabolomics: Application in Plant Sciences -- 13.1 Introduction -- 13.2 Sample Preparation -- 13.2.1 Culture and Harvesting -- 13.2.2 Storage and Drying -- 13.2.3 Extraction -- 13.3 Analytical Methods.

13.3.1 NMR-Based Methods.