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Title Methodologies for metabolomics : experimental strategies and techniques / edited by Norbert W. Lutz, Jonathan V. Sweedler, Ron A. Wevers
Published Cambridge ; New York : Cambridge University Press, 2013
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Description 1 online resource (xii, 627 pages)
Contents Contributors -- Section 1 Basic Methodological Strategies in Metabolomic Research -- 1 Exploring the Human Metabolome by Nuclear Magnetic Resonance Spectroscopy and Mass Spectrometry -- 1. Brief History of Human Metabolomics -- 2. Sample Preparation for Human Metabolomic Studies -- 3. Human Metabolite Identification by Nuclear Magnetic Resonance Spectroscopy -- 4. Human Metabolite Identification by Gas Chromatography-Mass Spectrometry -- 5. Human Metabolite Identification by Liquid Chromatography-Mass Spectrometry -- 6. Comparison of Technologies -- 7. Conclusion -- Acknowledgments -- 2 Methodological Requirements for Lipidomics Research -- 1. Sample Preparation and Lipid Extraction -- 2. Sample Preparation -- 3. Lipid Extraction -- 4. Sample Preparation and Lipid Extraction of Subcellular Organelles -- 5. Chromatography-Coupled Mass Spectrometry of Lipids -- 6. Shotgun Lipidomics -- Direct Infusion-Based Mass Spectrometry of Lipids -- 7. Postacquisition Data Processing and Bioinformatics -- 8. Conclusion -- Acknowledgments -- 3 Biological Methods for Metabolic Research -- 1. Gene Overexpression as a Tool for Metabolomic Studies -- 1.1. Gene Transfection: Plasmid Vectors -- 1.2. Gene Transduction: Viral Vectors -- 2. Gene Silencing as a Tool for Metabolomic Studies -- 3. Validation of Gene Overexpression or Silencing by mRNA -- 3.1. Gene Expression Analysis -- 3.1.1. Reverse Transcriptase Polymerase Chain Reaction -- 3.1.2. Real-Time Polymerase Chain Reaction -- 4. Validation of Gene Overexpression or Silencing by Protein Level Analysis and Enzyme Assays -- 4.1. Western Blotting -- 4.1.1. Blotting -- 4.1.2. Immunodetection -- 4.2. Enzyme Assays -- 4.2.1. Protocols for Analysis of Choline Kinase- Activity -- 4.2.2. Protocol for Analysis of Phospholipase D Activity
5. Enzymatic Assays as a Tool of Phospholipid Metabolism Analysis: Effects of Choline Kinase- Inhibition on the Balance of Phospholipid Metabolites -- 5.1. Protocol for Lipid Analysis -- 5.2. Protocol for Analysis of Phosphatidylinositol-3-Kinase Activity -- 6. Phospholipid Profile Analysis by Mass Spectrometry -- 6.1. Phospholipid Extraction -- 6.2. Analytical Technique for Phospholipids: Mass Spectrometry -- Section 2 Metabolomic Mass Spectrometry: Experimental Techniques and Bioinformatics -- 4 Considerations in Sample Preparation, Collection, and Extraction Approaches Applied in Microbial, Plant, and Mammalian Metabolic Profiling -- 1. Considerations in the Culturing, Collection, Preparation, and Extraction of Microbial Samples -- 1.1. Sample Types -- 1.2. Culture Methods -- 1.2.1. Batch Growth -- 1.2.2. Continuous Culture -- 1.2.3. Selection of Media -- 1.3. Experimental Considerations in the Collection and Preparation of Samples -- 1.3.1. Quenching of Cultures -- 1.3.2. Assessment of Leakage -- 1.3.3. Extraction of Intracellular Metabolites -- 2. Considerations in the Growth, Harvest, Preparation, and Extraction of Plant Materials -- 2.1. Considerations in Experimental Design and Growth of Plant Material -- 2.2. Plant Material Collection -- 2.3. Preparation and Storage of Plant Sample Material -- 2.4. Extraction of Plant Materials for High-Throughput Nontargeted Metabolic Profiling -- 2.4.1. Considerations for Plant Intracellular Metabolite Extraction -- 2.4.2. Reference Protocols Applied to Intracellular Metabolite Extraction in Plant Metabolic Profiling -- 3. Considerations in the Preparation, Collection, and Extraction of Clinical and Mammalian Samples -- 3.1. Sample Types and Considerations in Choice of Sample -- 3.1.1. Study Environment -- Controlled Laboratory versus the General Population -- 3.1.2. Biological Study Objectives
3.1.3. Sample Collection and Preparation Issues -- 3.2. Sample Collection and Sample Preparation -- 3.2.1. Urine -- 3.2.2. Serum and Plasma -- 3.2.3. Cell Line Cultures -- Intracellular and Metabolic Footprint -- 3.2.4. Tissues -- 4. General Considerations -- 4.1. Internal Standards -- 4.2. Sample Processing and Preparation for Analysis Following Extraction -- 4.3. Use of Quality Control and Blank Samples to Monitor Technical and Analytical Error and to Provide High-Quality Metabolic Profiling Data -- 5. Conclusion -- Acknowledgments -- 5 Mass Spectrometry-Based Methodologies for Single-Cell Metabolite Detection and Identification -- 1. Methodologies: Overview and Timing -- 2. Materials -- 2.1. Sample Preparation -- 2.1.1. Animals -- 2.1.2. Reagents and Solutions -- 2.1.3. Disposables -- 2.1.4. Equipment -- 2.2. Capillary Electrophoresis-Electrospray Ionization Mass Spectrometry System -- 2.2.1. Reagents and Solutions -- 2.2.2. Equipment -- 2.2.3. Software -- 2.3. Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry System -- 2.3.1. Reagents and Solutions -- 2.3.2. Equipment -- 2.3.3. Software -- 3. Sample Preparation -- 3.1. Single Cell Isolation -- 3.1.1. Sample Preparation for Capillary Electrophoresis-Electrospray Ionization Mass Spectrometry -- 3.1.2. Sample Preparation for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry -- 3.2. Instrument System Setup -- 3.2.1. Capillary Electrophoresis-Electrospray Ionization Mass Spectrometry -- 3.2.2. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry -- 3.3. Single-Cell Capillary Electrophoresis-Electrospray Ionization Mass Spectrometry Analysis -- 3.4. Single-Cell Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Analysis -- 3.5. Data Interpretation -- 3.5.1. Capillary Electrophoresis-Electrospray Ionization Mass Spectrometry Results
3.5.2. Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Results -- 4. Exemplary Applications in Single-Cell Mass Spectrometry -- 4.1. Metabolomics with Single-Cell Capillary Electrophoresis-Electrospray Ionization Mass Spectrometry -- 4.2. Metabolomics with Single-Cell Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry -- 5. Conclusion -- Acknowledgments -- 6 Direct Metabolomics from Tissues and Cells: Laser Ablation Electrospray Ionization for Small Molecule and Lipid Characterization -- 1. Direct Tissue Analysis and Imaging with Laser Ablation Electrospray Ionization -- 1.1. Label-Free Metabolite Identification -- 1.2. Metabolic Analysis -- 1.3. Metabolic Imaging -- 1.4. Metabolic Depth Profiling and Three-Dimensional Imaging -- 2. Metabolic Analysis of Single Cells -- 7 Bioinformatic Approaches to Processing and Annotation of High-Resolution Mass Spectrometry Data -- 1. Materials -- 1.1. Preparation and Metabolite Extraction of Biological Samples -- 1.1.1. Laboratory Instrumentation -- 1.1.2. Consumables -- 1.2. Fourier Transform Ion Cyclotron Resonance Mass Spectrometry -- 1.3. Data Management and Software -- 2. Methods -- 2.1. Preparation and Metabolite Extraction of Biological Samples -- 2.2. Fourier Transform Ion Cyclotron Resonance Mass Spectrometry -- 2.2.1. Selective Ion Monitoring Stitching -- 2.2.2. Measurement of Mass Spectra -- 2.3. From Raw Spectral Data to Processed Mass Spectra -- 2.3.1. Transient Summation -- 2.3.2. Apodization -- 2.3.3. Zero-Filling -- 2.3.4. Fast Fourier Transform -- 2.3.5. Peak Detection and Quantification -- 2.3.6. External and Internal Calibration -- 2.3.7. Selective Ion Monitoring Stitching Algorithm -- 2.3.8. Signal Filtering of Processed Mass Spectra -- 2.4. Putative Nonstructural Identification of Mass Measurements -- 2.4.1. Theoretical Background
2.4.2. Empirical Formulae Assignments Including Heuristic Rules -- 2.4.3. Filtering Assignments of Empirical Formulae Using Relative Isotopic Abundances -- 2.5. Putative Structural Identification of Mass Measurements -- 2.5.1. Theoretical Background -- 2.5.2. Single-Peak Search Using a Modified Kyoto Encyclopedia of Genes and Genomes Database -- 2.5.3. Peak Assignment Using Prior Biological Knowledge -- Acknowledgments -- 8 Approaches for Natural Product Detection and Structural Elucidation Using Mass Spectrometry with High Mass Accuracy -- 1. Materials -- 1.1. Bacterial Strains -- 1.2. Solvents and Buffers -- 1.3. Instrumentation -- 2. Methods -- 2.1. Sample Preparation -- 2.2. Liquid Chromatography Methods -- 2.2.1. Reverse Phase Liquid Chromatography -- 2.2.2. Hydrophilic Interaction Liquid Chromatography -- 2.3. Mass Spectrometry Methods -- 2.3.1. Fourier-Transform Mass Spectrometry Detection of Metabolites -- 2.3.1.1. Targeted Detection of Metabolites and Quantitation. -- 2.3.2. Fragmentation Methods -- 2.3.2.1. Collision-Induced Dissociation. -- 2.3.2.2. Pulsed Q Dissociation. -- 2.3.2.3. Targeted Fragmentation. -- 2.3.2.4. Data-Dependent Fragmentation. -- 2.4. Data Analysis -- 2.4.1. Empirical Formula Determination -- 2.4.2. Fragmentation Patterns -- Acknowledgments -- 9 Metabolomics Using Ion Mobility Mass Spectrometry -- 1. Overview -- 2. Ambient Pressure Ion Mobility Mass Spectrometry -- 2.1. Introduction to Drift Tube Ion Mobility Mass Spectrometry -- 2.2. Data Collection and Processing -- 3. Traveling Wave Ion Mobility Mass Spectrometry -- 3.1. Sample Introduction and Ionization -- 3.2. Traveling Wave Ion Mobility Spectrometry -- 3.3. Data Collection and Processing -- 4. Protocol for Metabolomics Applications Using Ion Mobility Mass Spectrometry -- 4.1. Measuring Metabolomes by Ion Mobility Mass Spectrometry -- 4.2. Comparing Metabolomes
Summary "While most of the focus in "omics" science over the past decade has been on sequencing the human genome [1] or annotating the human proteome [2], there is another equally important component of the human body that has, until recently, been largely overlooked: the human metabolome. The human metabolome can be thought of as the complete collection of small molecule metabolites found in our bodies. These small molecules include such chemical entities as peptides, amino acids, nucleic acids, carbohydrates, organic acids, vitamins, minerals, food additives, drugs and just about any other chemical (with a molecular weight 1500 Da) that can be used, ingested or synthesized by humans. Metabolites act as the bricks and mortar of our cells. They serve as the building blocks for all of our macromolecules including proteins, RNA, DNA, carbohydrates, membranes and all other biopolymers that give our cells their structure and integrity. Metabolites also act as the fuel for all cellular processes, the buffers to help tolerate environmental insults and the messengers for most intra- and intercellular events. Together with the genome and the proteome, the human metabolome essentially defines who and what we are"--Provided by publisher
Bibliography Includes bibliographical references and index
Notes Online resource; title from digital title page (viewed on Feb. 25, 2013)
Subject Mass spectrometry
Metabolites -- Analysis
Molecular diagnosis
Metabolism -- Disorders
Metabolomics -- methods
Mass Spectrometry -- methods
Metabolic Diseases -- diagnosis
Metabolome
SCIENCE -- Chemistry -- Analytic.
Mass spectrometry.
Metabolism -- Disorders.
Molecular diagnosis.
Form Electronic book
Author Lutz, Norbert, 1952-
Sweedler, Jonathan V
Wevers, Ron, 1951-
LC no. 2012013907
ISBN 9780511996634 (electronic bk.)
0511996632 (electronic bk.)
9781139611619 (e-book)
1139611615