Preface
Contents
Contributors
Chapter 1: Methods for the Development of Recombinant Microorganisms for the Production of Natural Products
1 Introduction
2 Methods
2.1 Static Balancing
2.2 Dynamic Balancing
2.3 Co-culturing
2.4 Fermentation Optimization
2.5 Media Optimization
2.6 Temperature Optimization
2.7 Induction Optimization
References
Chapter 2: Sustainable Technological Methods for the Extraction of Phytochemicals from Citrus Byproducts
1 Introduction
2 Materials
2.1 Ultrasound-Assisted Extraction
2.2 Enzyme-Assisted Extraction
2.3 Head Space Solid Phase Microextraction (HS-SPME)
2.4 Fermentation with Lactic Acid Bacteria
3 Methods
3.1 Ultrasound-Assisted Extraction
3.2 Enzyme-Assisted Extraction
3.3 Head Space Solid Phase Microextraction (HS-SPME)
3.4 Fermentation with Lactic Acid Bacteria
4 Notes
References
Chapter 3: Reconstitution of Metabolic Pathway in Nicotiana benthamiana
1 Introduction
2 Materials
2.1 Materials and Reagents
2.2 Equipment
2.3 Materials for UPLC/MS Analyses
3 Methods
4 Notes
References
Chapter 4: A Protocol for Phylogenetic Reconstruction
1 Introduction
2 Materials
2.1 The Following Programs Need to Be Downloaded and Installed on a Local Computer
2.2 Data
3 Methods
3.1 Selection of Candidate Genes Using PSI-BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi)
3.2 Domain Analysis Using PFAM (https://pfam.xfam.org/)
3.3 Multiple Sequence Alignment (MSA) Using MUSCLE
3.4 MSA Gap Treatment Using trimAL (http://trimal.cgenomics.org/)
3.5 Selection of Best-Fit Models of Protein Evolution Using ProtTest (https://github.com/ddarriba/ProtTest3/releases)
3.6 Maximum Likelihood Analysis Using PhyML (http://www.atgc-montpellier.fr/PhyML/)
3.7 Tree Visualization Using FigTree
3.8 Phylogenetic Tree of Plant UGTs and Visualization Using iToL
4 Notes
References
Chapter 5: RNA-Seq Data Analysis Pipeline for Plants: Transcriptome Assembly, Alignment, and Differential Expression Analysis
1 Introduction
2 Materials
2.1 Software and Data
3 Methods
3.1 Read Preparation and Quality Assessment
3.1.1 Initial Quality Check
3.1.2 Adapter Trimming and Adapter Removal
3.2 De Novo Transcriptome Assembly Using Trinity
3.3 RNA-Seq Alignment at the Gene and Transcript Level
3.3.1 Genome Indexing and Alignment Using STAR
3.4 Transcript Indexing and Alignment Using Salmon
3.5 Data Preparation for DeSEQ2 Input
4 Notes
References
Chapter 6: A Protocol for Horizontally Acquired Metabolic Gene Detection in Algae
1 Introduction
2 Materials
2.1 Programs and Data
3 Methods
4 Note
References
Chapter 7: Global Comparative Label-Free Yeast Proteome Analysis by LC-MS/MS After High-pH Reversed-Phase Peptide Fractionatio...
1 Introduction
2 Materials
2.1 Common Laboratory Consumables
2.2 Growth Conditions, Treatment, Sample Collection, and Sample Preparation
2.3 Protein Extraction, Reduction, Alkylation, and Digestion
2.4 High-pH Reversed-Phase Fractionation
2.5 Data-Dependent NanoLC-MS/MS Analyses
2.6 Data Processing, Statistical Analysis, and Bioinformatics
3 Methods
3.1 Cell Growth
3.2 Sample Collection and Processing
3.3 Protein Extraction, Reduction, Alkylation, and Digestion
3.4 Solid Phase Extraction with SOLA High-pH Fractionation
3.5 Data-Dependent NanoLC-MS/MS Analyses
3.6 Data Processing and Statistical Analysis
3.7 Bioinformatics
4 Notes
References
Chapter 8: Gas Chromatography Coupled to Atmospheric Pressure Chemical Ionization High-Resolution Mass Spectrometry for Metabo...
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
3 Methods
3.1 Extraction and Derivatization of Grape Berry Metabolites
3.2 Derivatization of Commercial Standards
3.3 GC Parameters
3.4 APGC-MSE Parameters
3.5 Data Processing
4 Notes
References
Chapter 9: GC-MS/MS Profiling of Plant Metabolites
1 Introduction
2 Materials
2.1 Sample Preparation and Metabolite Extraction
2.2 Sample Derivatization
2.3 GC-MS Instrumentation
2.4 GC-MS Analysis
2.5 Data Analysis
3 Methods
3.1 SRM Method Development Using AutoSRM Software
3.2 Authentic Standard Compound Preparation
3.3 Sample Derivatization
3.4 GC-MS Analysis
3.5 Precursor Ion Selection
3.6 Product Ion Selection
3.7 Collision Energy Optimization
3.8 Generating Master Compound Database
3.9 Creating Analysis Method
3.10 Arabidopsis Sample Preparation
3.11 Metabolite Extraction
3.12 Sample Derivatization and GC-MS Analysis
References
Chapter 10: Analysis of Grape Volatiles Using Atmospheric Pressure Ionization Gas Chromatography Mass Spectrometry-Based Metab...
1 Introduction
2 Materials
2.1 Sample Collection
2.2 Chemicals and Standards
2.3 Sample Preparation and Extraction Equipment
2.4 APGC-TOF-MS Instrumentation
2.5 Data Analysis Software
3 Methods
3.1 Volatile Metabolite Extraction from Grape Berries
3.2 Solid-Phase Extraction of Volatile Compounds
3.3 APGC-TOF-MS Analysis
3.4 Development of APGC-HRMS Spectral Library
3.5 Data Analysis
4 Notes
References
Chapter 11: A High-Throughput HILIC-MS-Based Metabolomic Assay for the Analysis of Polar Metabolites
1 Introduction
2 Materials
2.1 Equipment
2.2 Solvents and Chemicals
2.3 Supplies
3 Methods
3.1 Sample Preparation
3.2 LC-MS Analysis of Polar Metabolites
3.3 LC Conditions
3.4 MS Conditions
3.5 Cleaning Procedures
3.6 Untargeted Data Processing and Analysis
3.7 Method Validation
References
Chapter 12: Macrolipidomic Profiling of Vegetable Oils: The Analysis of Sunflower Oils with Different Oleic Acid Content
1 Introduction
2 Materials
2.1 Lipid Extraction Solvents
2.2 Dual Serial UHPLC Column Coupling and Mobile Phase Preparation
2.3 Mass Spectrometry and Processing Software
3 Methods
3.1 Lipid Extraction, Dilution, and Reconstitution
3.2 Instrument Setup and Equilibration, Method Settings
3.3 Data Acquisition
3.4 Instrument Flush
3.5 Data Processing
4 Notes
References
Chapter 13: Non-targeted Lipidomics Using a Robust and Reproducible Lipid Separation Using UPLC with Charged Surface Hybrid Te...
1 Introduction
2 Materials
2.1 Chemicals and Lipid Standards
2.2 UPLC-MS Equipment
2.3 Other Instrumentation
2.4 Data Processing
3 Methods
3.1 Lipid System Suitability Standard Preparation
3.2 Plasma Total Lipid Extraction
3.3 LC Mobile Phase and Wash Solution Preparation
3.4 LC-MS Nontargeted Platform
3.5 Data Processing
4 Notes
References
Chapter 14: Comprehensive Analysis of Plant Lipids Using Sub-2-ฮผm Particle CO2-Based Chromatography Coupled to Mass Spectromet...
1 Introduction
2 Materials
2.1 Sample Preparation
2.2 SFC-MS/MS Analysis
2.3 Data Analysis
3 Methods
3.1 Lipid Extraction
3.2 Optimizing MS Conditions and Chromatographic Separation for Different Lipid Species
3.3 SFC-ESI-MSE Analysis of Plant Lipids
3.4 Data Processing
3.5 Lipid Identification
4 Notes
References
Chapter 15: Bioinformatics in Lipidomics: Automating Large-Scale LC-MS-Based Untargeted Lipidomics Profiling with SimLipid Sof...
1 Introduction
2 Materials
3 Methods
3.1 Implementation
3.2 Database
3.3 SimLipid Data Analysis Workflow
3.4 Preliminary Data Processing
3.5 LC-MS Data Processing
3.6 Data-Dependent Acquisition (DDA) Method
3.7 Data-Independent Acquisition (DIA) Method
3.8 Retention Time Alignment
3.9 Create LC-MS Template
3.10 Lipid Identification
3.11 Data Normalization Using Internal Standards
3.12 Portable Reports and Statistical Analysis
3.13 Example on Performance Evaluation of SimLipid Software Using Large-Volume LC-MS and MS/MS Dataset
4 Notes
References
Chapter 16: A Protocol for Prion Discovery in Plants
1 Introduction
2 PLAAC
3 Application of Hidden Markov Models
4 Limitations of the PrLD HMM
5 Use of PLAAC on the Web
6 Methods
References
Chapter 17: Structural Determination of Uridine Diphosphate Glycosyltransferases Using X-Ray Crystallography
1 Introduction
2 Materials
3 Methods
3.1 Protein Expression and Purification
3.2 SeMet Protein Preparation
3.3 Crystallization
3.4 X-Ray Diffraction Data Collection
3.4.1 UGT85H2
3.4.2 UGT71G1
3.5 Structure Determination and Refinement
3.5.1 UGT71G1: Multiwavelength Anomalous Dispersion (MAD)
3.5.2 UGT85H2: Molecular Replacement
3.5.3 Structure Validation
3.6 Structural Analysis
3.6.1 Overall Structure
3.6.2 Interactions between Enzyme and Donor Ligands
3.6.3 The Acceptor Binding Site
3.7 Molecular Docking
References
Index