Plant Lipids

Preface
Contents
Contributors
Part I: Basic Methods of Lipid Isolation and Analysis
	Chapter 1: Lipid Isolation from Plants
		1 Introduction
		2 Materials
			2.1 Preparing Solvents
			2.2 Harvesting Tissue and Storage
			2.3 Tissue Homogenization
			2.4 Lipid Extraction
			2.5 Quantification of Total Lipids
		3 Methods
			3.1 Preparation of Solvents (Protocol Designed for Samples of 1 g or Less)
			3.2 Harvesting Tissue for Storage
			3.3 Tissue Homogenization
			3.4 Lipid Extraction
			3.5 Quantification of Total Lipids by GC Analysis of Fatty Acid Methyl Esters
		4 Notes
		References
	Chapter 2: Three Methods to Extract Membrane Glycerolipids: Comparing Sensitivity to Lipase Degradation and Yield
		1 Introduction
		2 Materials
			2.1 Solvents
			2.2 Laboratory Equipment
		3 Methods
			3.1 Plant Tissue Collection
			3.2 Extraction Method (Adapted from Wang and Benning, 2011)
			3.3 Extraction Method (Adapted from J. A. Browse; See Chapter 1)
			3.4 Extraction Method (Adapted from Shiva et al., 2018)
		4 Notes
		References
	Chapter 3: Thin-Layer Chromatography
		1 Introduction
		2 Materials
			2.1 One-Dimensional Thin-Layer Chromatography (1D-TLC)
			2.2 Two-Dimensional Thin-Layer Chromatography (2D-TLC)
			2.3 Reference (Standard) Lipids and Staining and Visualization of Lipids
		3 Methods (See Note 7)
			3.1 Lipid Extraction from Plant Tissues for TLC Separation
			3.2 Separation of Polar Lipids Via 1D-TLC (Fig. 1)
			3.3 Separation of Non-Polar Lipids Via 1D-TLC (Fig. 2)
			3.4 Separation of Polar Lipids Via 2D-TLC (Fig. 3) (See Note 10)
			3.5 Staining of Lipids with Iodine
			3.6 Staining of Lipids with Anilino Naphthalene Sulfonic acid (ANS)
			3.7 Staining of Lipids with Primuline
			3.8 Staining of Glycolipids with α-Naphthol-Sulfuric Acid
			3.9 General Lipid Staining by Charring with Sulfuric Acid [10, 11  ]
			3.10 Recovery of Lipids from TLC Plates
		4 Notes
		References
	Chapter 4: Lipid Analysis by Gas Chromatography and Gas Chromatography-Mass Spectrometry
		1 Introduction
		2 Materials
			2.1 Materials for Lipid Derivatization
			2.2 Internal Standards
			2.3 Gas Chromatography
		3 Methods
			3.1 Preparation of FAMEs by Transmethylation and Quantification by GC-FID
			3.2 Trimethylsilyation of Long Chain Alcohols and Quantification by GC-MS
			3.3 Trimethyl-silylation and GC-MS Analysis of Monoacylglycerols
			3.4 Analysis of Double Bond Position in Unsaturated Fatty Acids by GC-MS
		4 Notes
		References
	Chapter 5: 14C-Tracing of Lipid Metabolism
		1 Introduction
			1.1 Stage of Seed Development
			1.2 Choice of Radiolabeled Substrate
			1.3 Type of Labeling Experiment and Time Points
			1.4 Variations in Structural Labeling Aid in Defining Pathway Flux
		2 Materials
			2.1 Seed Staging and Collection
			2.2 Continuous Labeling Experiment
			2.3 Lipid Extraction
			2.4 Lipid Analysis
			2.5 14C Labeled Lipid Regiochemical Analysis
		3 Methods
			3.1 Developing A. thaliana Seed Staging and Collection
			3.2 Continuous Labeling Experiment
			3.3 Lipid Extraction
			3.4 Analysis of the 14C Labeled Lipid Extract by Thin-Layer Chromatography
			3.5 Analysis of the 14C Labeled Lipid Extract High-Performance Liquid Chromatography
			3.6 Elution of Lipids from Silica Gel
			3.7 Regiochemical Analysis of 14C Labeled TAG and DAG
			3.8 Regiochemical Analysis of 14C Labeled PC
		4 Notes
		References
	Chapter 6: Methods of Lipid Analyses for Microalgae: Charophytes, Eustigmatophytes, and Euglenophytes
		1 Introduction
		2 Materials
			2.1 Algal Strains
			2.2 Medium and Algal Growth
			2.3 Cell Harvesting and Lipid Extraction
			2.4 Thin-Layer Chromatography (TLC)
			2.5 Derivatization and Gas Chromatography (GC)
		3 Methods
			3.1 Preparation of Cells
				3.1.1 Cell Growth for Membrane Lipid Analysis
				3.1.2 Growth of K. nitens for Surface Lipid Analysis
			3.2 Extraction of Lipids
				3.2.1 Extraction of Membrane and Neutral Lipids
				3.2.2 Chloroform Extraction of Surface Lipids from K. nitens
				3.2.3 Silica Gel Extraction of Surface Lipids from K. nitens
			3.3 Separation of Lipids
				3.3.1 2D-TLC Separation of Membrane Lipids
				3.3.2 Two-Way TLC Separation of Membrane Lipids
				3.3.3 TLC Separation of Neutral Lipids (TAGs and Wax Esters)
				3.3.4 TLC Separation of Surface Lipids (Alkanes, Steryl Esters, Phytyl Esters, Sterols)
			3.4 Derivatization of Lipids
				3.4.1 Preparation of FAMEs
				3.4.2 Derivatization of Sterols
				3.4.3 Derivatization of Steryl Esters
			3.5 Gas Chromatography (GC) Analysis
		4 Notes
		References
Part II: Mass Spectrometry and NMR Analysis
	Chapter 7: Direct Infusion Mass Spectrometry for Complex Lipid Analysis
		1 Introduction
		2 Materials
			2.1 Lipid Extraction and Solid-Phase Extraction
			2.2 Lipid Standards for Q-TOF MS/MS
			2.3 Direct Infusion MS/MS and Data Analysis
		3 Methods
			3.1 Lipid Extraction with Phospholipase Inactivation Using Boiling Water
			3.2 Lipid Fractionation Via SPE
			3.3 Direct Infusion MS/MS of Plant Lipids
			3.4 Data Analysis of Direct Infusion MS/MS Experiments
		4 Notes
		References
	Chapter 8: Fatty Acid Composition by Total Acyl Lipid Collision-Induced Dissociation Time-of-Flight (TAL-CID-TOF) Mass Spectro...
		1 Introduction
		2 Materials
		3 Methods
			3.1 Sample Preparation and Mass Spectrometry (Waters Mass Spectrometers) (See Note 2)
			3.2 Mass Spectrometry (MDS/Sciex QStar Elite)
			3.3 LipidomeDB Data Calculation Environment (DCE)
		4 Notes
		References
	Chapter 9: Targeted Analysis of the Plant Lipidome by UPLC-NanoESI-MS/MS
		1 Introduction
		2 Materials
			2.1 Samples and Buffers
			2.2 Chemicals and Standards
			2.3 Solvents and Solutions for LC-MS
			2.4 LC-MS System
			2.5 Software
			2.6 Other Equipment
		3 Methods
			3.1 Harvesting and Homogenization of Plant Material
			3.2 Enrichment of Microsomal Membrane Fractions
			3.3 Extraction of Lipids from Plant Material and Cultured Cells
			3.4 Extraction of Lipids from Microsomal Membrane Fractions
			3.5 Chemical Derivatization of Lipids
			3.6 Methylamine Treatment for Enhanced Sphingolipid Analysis
			3.7 Lipid Analysis by UPLC-NanoESI-Mass Spectrometry
			3.8 Assembly of the Lipid Building Blocks for the Target Lipid List
			3.9 Data Analysis and Processing
			3.10 Absolute Quantification of Lipid Subclasses by TLC Coupled with GC-FID
		4 Notes
		References
	Chapter 10: Mass Spectrometry-Based Profiling of Plant Sphingolipids from Typical and Aberrant Metabolism
		1 Introduction
		2 Materials
			2.1 Lipid Extraction
			2.2 UPLC and Mass Spectrometry
			2.3 Sphingolipid Standards
		3 Methods
			3.1 Sphingolipid Extraction
			3.2 UPLC-MS Detection of Sphingolipids
			3.3 Updates for Profiling of Aberrant Sphingolipids
				3.3.1 Variant Products of Serine Palmitoyltransferase Activity
				3.3.2 Glucosylceramides with Nonhydroxylated Fatty Acids
				3.3.3 Variations in GIPC Classes
		4 Notes
		References
	Chapter 11: Analysis of Free and Esterified Sterol Content and Composition in Seeds Using GC and ESI-MS/MS
		1 Introduction
		2 Materials
			2.1 Seed Oil Extraction
			2.2 Saponification of Lipid Extracts and Sterol Silylation for GC Analysis
			2.3 Free Phytosterol Derivatization with Undecanoyl Chloride
			2.4 Semi-preparative Purification of Steryl Esters
			2.5 Direct Infusion ESI-MS/MS of Steryl Ester Fraction
		3 Methods
			3.1 Seed Oil Extraction
			3.2 Saponification of Lipid Extracts and Sterol Silylation for GC-FID/MS Analysis
			3.3 Free Phytosterol Derivatization with Undecanoyl Chloride
			3.4 Semipreparative Purification of Steryl-Esters
			3.5 Direct Infusion ESI-MS/MS Analysis of Steryl Ester Fractions
			3.6 Data Processing
		4 Notes
		References
	Chapter 12: Techniques for the Measurement of Molecular Species of Acyl-CoA in Plants and Microalgae
		1 Introduction
		2 Materials
			2.1 Acyl-CoA Standards
			2.2 Preparation of Chloroacetaldehyde Derivitization Solution
			2.3 Acyl-CoA Extraction
			2.4 Separation of Acyl-CoA Species Using Liquid Chromatography and Ultraviolet (UV) Detection
			2.5 Separation of Derivatized Acyl-CoA Species Using Liquid Chromatography and Fluorescence Detection
			2.6 Separation of Isobaric Short Chain Acyl-CoA Species (Derivatized or Not) Using Liquid Chromatography
			2.7 Separation of Acyl-CoA Species Using Liquid Chromatography and Analysis by Mass Spectrometry with Multiple Reaction Monito...
		3 Methods
			3.1 Acyl-CoA Standards
			3.2 Preparation of Chloroacetaldehyde Derivatization Solution
			3.3 Acyl-CoA Extraction
			3.4 Analysis of Nonderivatized Acyl-CoA Thioesters Using Liquid Chromatography with Ultraviolet Detection
			3.5 Separation and Analysis of Derivatized Acyl-CoA Species Using Liquid Chromatography and Fluorescence Detection
			3.6 Separation of Acyl-CoA Species Using Liquid Chromatography and Analysis by Tandem Mass Spectrometry with MRM
			3.7 Diagnostic Ions for the Identification of Acyl-CoA Species and Data Processing
		4 Notes
		References
	Chapter 13: Quantification of Acyl-Acyl Carrier Proteins for Fatty Acid Synthesis Using LC-MS/MS
		1 Introduction
		2 Materials
			2.1 General Material and Equipment
			2.2 Overexpression of apo-ACP and Sfp Transferase in E. coli and Purification
			2.3 Acyl-ACP Standard Synthesis
			2.4 Analysis of Acyl-ACPs by SDS-PAGE
			2.5 Extraction of Acyl-ACP Standards
			2.6 Extraction of Acyl-ACP from Plant Tissues
			2.7 Endoproteinase Asp-N Digestion
			2.8 LC-MS/MS
			2.9 Software
		3 Methods
			3.1 Overexpression of apo-ACP and Sfp Transferase in E. coli and Harvesting of Cells
			3.2 Purification of apo-ACP and Sfp Transferase by Ni2+ IMAC Chromatography
			3.3 Concentration and Desalting of Recombinant apo-ACP and Sfp Transferase
			3.4 Acyl-ACP Standard Synthesis
			3.5 Making the Calibration Curve for Quantification of Acyl-ACPs
				3.5.1 Single Point Quantification for Estimation
				3.5.2 15N Labeled Standard Mix/15N Standard Diluent
				3.5.3 Unlabeled Standard Mix
				3.5.4 Serial Dilution
				3.5.5 Example Standard Curve Generation
			3.6 SDS-PAGE Analysis of Synthesized Acyl-ACP Standards
			3.7 Clean-Up of Acyl-ACP Standards by TCA Extraction
			3.8 Acyl-ACP Extraction from Plant Tissues
			3.9 Endoproteinase Asp-N Digestion of Acyl-ACPs and Sample Clean-Up
			3.10 LC-MS/MS Analysis
			3.11 LC-MS/MS Data Analysis and Quantification
		4 Notes
		References
	Chapter 14: Structural Analysis of Glycosylglycerolipids Using NMR Spectroscopy
		1 Introduction
		2 Materials
			2.1 NMR Tubes and Solvents
			2.2 Lipids Analyzed
		3 Methods
			3.1 Sample Preparation
			3.2 NMR Experiments
			3.3 Interpretation Workflow
		4 Notes
		References
Part III: Lipid Isolation and Analysis from Plant Tissues Cell Compartments and Organelles
	Chapter 15: Analysis of Extracellular Cell Wall Lipids: Wax, Cutin, and Suberin in Leaves, Roots, Fruits, and Seeds
		1 Introduction
		2 Materials
			2.1 Glassware, Syringes, Laboratory Tools
			2.2 Solvents for Lipid Extraction and Sample Preparation
			2.3 Reagents for Depolymerization, Transesterification and Derivatization
			2.4 Standards for GC-FID and GC-MS Analysis
			2.5 GC-FID and GC-MS Instruments
		3 Methods
			3.1 Collection and Preparation of Plant Samples
			3.2 Enzymatic Isolation of Extracellular Lipid Polymers
			3.3 Wax Extraction from Plant Organs and Cutin or Suberin Polymers and Preparation of Wax-Free Cuticular Membranes
			3.4 Mechanical Isolation of Epicuticular Wax Using Collodion
			3.5 Wax Extraction from One Leaf Side Using Glass Vials with Rolled Edges
			3.6 Depolymerization and Transesterification of Cutin and Suberin Using Methanolic HCl
			3.7 Depolymerization and Transesterification of Suberin and Cutin Using Boron Trifluoride-Methanol
			3.8 Derivatization with BSTFA
			3.9 Preparation of Internal Standards
			3.10 GC-FID and GC-MS
			3.11 Column Maintenance and Acid Standard Chromatography
		4 Notes
		References
	Chapter 16: Isolation of Lipid Droplets for Protein and Lipid Analysis
		1 Introduction
		2 Materials
			2.1 General Lab Supplies
			2.2 LD Isolation
			2.3 LD Lipid Extraction and Analysis (See Note 3)
			2.4 Protein Processing
			2.5 Proteomic Analysis
			2.6 Verification of Protein-LD Localization by Expression in N. benthamiana Leaves
			2.7 Verification of LD Localization by Expression in N. tabacum Pollen Tubes
		3 Methods
			3.1 Sample Collection and LD Isolation
			3.2 Lipid Extraction from LDs
			3.3 Lipid Separation by TLC
			3.4 Lipid Extraction from TLC Plates
			3.5 Preparation of Fatty Acid Methyl Esters (FAMEs) and GC Analysis
			3.6 Protein Isolation, Gel Electrophoresis, In-Gel Trypsin Digestion, and Proteomic Analysis by LC-MS
			3.7 Processing of LC-MS Data by MaxQuant
			3.8 Analysis of LC-MS Data by Perseus
			3.9 Verification of Protein Localization to LDs by Expression in N. benthamiana Leaves (Fig. 2)
			3.10 Verification of Protein Localization to LDs by Expression in N. tabacum Pollen Tubes (See Fig. 2)
		4 Notes
		References
	Chapter 17: Isolation of Plastoglobules for Lipid Analyses
		1 Introduction
		2 Materials
			2.1 Arabidopsis thaliana Cultivation
			2.2 Plastoglobule Purification by Flotation
			2.3 Plastoglobule Lipid Extraction
			2.4 Lipid Separation by Thin Layer Chromatography
			2.5 Quantification of Lipids After Synthesis and Analysis of Fatty Acid Methyl Esters by Gas Chromatography
		3 Methods
			3.1 Arabidopsis thaliana Cultivation (See Note 7)
			3.2 Plastoglobule Purification by Flotation
			3.3 Plastoglobule Lipid Isolation by Liquid-Liquid Extraction (See Note 21)
			3.4 Lipid Separation by Thin-Layer Chromatography
			3.5 Quantification of Lipids After Synthesis of Fatty Acid Methyl Esters and Analysis by Gas Chromatography
		4 Notes
		References
	Chapter 18: Isolation of Mitochondria for Lipid Analysis
		1 Introduction
		2 Materials
			2.1 Cell Cultures and Media
			2.2 Buffers and Equipment for Mitochondria Purification
			2.3 SDS-PAGE and Western Blot Analysis
		3 Methods
			3.1 Preparation of Cell Cultures
			3.2 Preparation of a Crude Mitochondria Fraction
			3.3 First Purification of Mitochondria on a Discontinuous Percoll Gradient
			3.4 Second Purification of Mitochondria on a Continuous Percoll Gradient
			3.5 Evaluation of Mitochondria Contamination by SDS-PAGE and Western Blot Analysis
			3.6 Lipid Analysis by Thin-Layer Chromatography or HPLC-MS/MS
		4 Notes
		References
	Chapter 19: Collection and Analysis of Phloem Lipids
		1 Introduction
		2 Materials
			2.1 Phloem Exudate Collection
			2.2 Phase Partitioning
			2.3 LC-ESI-MS
		3 Methods
			3.1 Harvest of Phloem Exudates (See Note 1)
			3.2 Purification/Enrichment of Phloem Lipids
			3.3 Lipid Analysis of Phloem Exudates
		4 Notes
		References
Part IV: Lipid Signaling, Lipid-Protein Interactions, and Imaging
	Chapter 20: Analyses of Inositol Phosphates and Phosphoinositides by Strong Anion Exchange (SAX)-HPLC
		1 Introduction
		2 Materials
			2.1 Plant Growth and Labeling of Inositol Polyphosphates
			2.2 Extraction of Inositol Polyphosphates
			2.3 High-Performance Liquid Chromatography (HPLC) of Inositol Polyphosphates
			2.4 Yeast Growth and Labeling of Phosphatidylinositol and Phosphoinositides
			2.5 Extraction and Deacylation of Phosphatidylinositol and Phosphoinositides
			2.6 High-Performance Liquid Chromatography (HPLC) of Deacylated Phosphatidylinositol and Phosphoinositides
		3 Methods
			3.1 Plant Growth and Steady-State Labeling of Inositol Polyphosphates
			3.2 Extraction of Inositol Polyphosphates
			3.3 Inositol Polyphosphate Analyses by HPLC
			3.4 Yeast Growth and Labeling of Phosphatidylinositol and Phosphoinositides
			3.5 Extraction and Deacylation of Phosphatidylinositol and Phosphoinositides
			3.6 HPLC Analyses of Deacylated Phosphatidylinositol and Phosphoinositides
		4 Notes
		References
	Chapter 21: Analysis of Phosphoinositides from Complex Plant Samples by Solid-Phase Adsorption Chromatography and Subsequent Q...
		1 Introduction
		2 Materials
			2.1 Acidic Extraction of Phosphoinositides (See Note 1)
			2.2 Separation and Enrichment of Lipid Classes by Solid-Phase Adsorption Chromatography
			2.3 Chromatographic Separation of Phosphoinositides
			2.4 Visualization of Lipid Standards on TLC Plates
			2.5 Derivatization of Phosphoinositide-Associated Fatty Acids
			2.6 GC Analysis of Fatty Acid Methyl Esters (FAMEs)
		3 Methods
			3.1 Acidic Extraction of Phosphoinositides
			3.2 Separation and Enrichment of Lipid Classes by Solid-Phase Adsorption Chromatography
			3.3 Chromatographic Separation of Phosphoinositides
			3.4 Visualization of Lipid Standards
			3.5 Isolation of Lipids from Silica Plates
			3.6 Transesterification of Lipid-Bound Fatty Acids for GC Analysis
			3.7 Analysis of Phosphoinositide-Associated Fatty Acids
			3.8 Data Analysis
		4 Notes
		References
	Chapter 22: Studying Lipid-Protein Interactions Using Protein-Lipid Overlay and Protein-Liposome Association Assays
		1 Introduction
		2 Materials
			2.1 Membrane Lipid Strips and Protein-Lipid Overlay Assay
			2.2 Preparation of Liposomes and Protein-Liposome Association Assay
		3 Methods
			3.1 Preparation of Membrane Lipid Strips for Protein-Lipid Overlay Assays
			3.2 Protein-Lipid Overlay Assay
			3.3 Preparation of Liposomes
			3.4 Protein-Liposome Association Assay
		4 Notes
		References
	Chapter 23: Investigations of Lipid Binding to Acyl-CoA-Binding Proteins (ACBP) Using Isothermal Titration Calorimetry (ITC)
		1 Introduction
		2 Materials
			2.1 Growth of Transformed Escherichia coli Cells Expressing the Recombinant Protein
			2.2 Protein Extraction and Purification
			2.3 Isothermal Titration Calorimetry (ITC)
		3 Methods
			3.1 Overexpression of His-Tagged Protein in E. coli and Protein Isolation
			3.2 Purification of His-Tagged Protein
			3.3 ITC
			3.4 ITC Data Analysis
		4 Notes
		References
	Chapter 24: In Situ Localization of Plant Lipid Metabolites by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry I...
		1 Introduction
		2 Materials
			2.1 Tissue Fixation (Optional)
			2.2 Tissue Embedding
			2.3 Tissue Cryo-Sectioning
			2.4 Matrix Application by Sublimation
			2.5 MALDI Mass Spectrometry
		3 Methods
			3.1 Sample Collection and Preservation
			3.2 Tissue Embedding
			3.3 Cryo-Sectioning
			3.4 Lyophilization
			3.5 Tissue Section Inspection and Bright-Field Microscopy
			3.6 Matrix Application by Sublimation
			3.7 MALDI-MS Imaging
			3.8 Data Processing
			3.9 Example Results
			3.10 Validation
		4 Notes
		References
Part V: Lipid Databases
	Chapter 25: Plant Lipid Databases
		1 Introduction
		2 Materials
		3 Methods
			3.1 Seed Oil Fatty Acids (SOFA) Database
			3.2 Plant Fatty Acid Database (PlantFAdb)
			3.3 Lipid Library
			3.4 Cyberlipid
			3.5 LipidWeb
			3.6 LipidBank Database
			3.7 PubChem
			3.8 LipidomicNet
			3.9 NIST Chemistry WebBook
			3.10 LipidHome
			3.11 Lipid Maps Lipidomics Gateway
			3.12 Metlin Database
		4 Notes
		References
	Chapter 26: Lipid Pathway Databases with a Focus on Algae
		1 Introduction
		2 Materials
		3 Methods
			3.1 General Comments
			3.2 General Homology Search for Identification of Orthologs: NCBI BLAST
			3.3 Detection of Orthologs in CyanoBase, MBGD, Ensembl, and  Pfam
			3.4 Ortholog Finding by Genome Clustering: Gclust
			3.5 Metabolite-Oriented Databases: CAZy and ARALIP
			3.6 Pathway Analysis with  KEGG
				3.6.1 Inferring Pathways in Organisms with Sequenced Genomes
				3.6.2 Mapping a Sequence of Interest onto a Pathway
				3.6.3 Detection of a Gene of Interest in Newly Sequenced Genomes
			3.7 Coexpression Analysis with ALCOdb
				3.7.1 Retrieval of Coexpressed Genes with a Single Guide  Gene
				3.7.2 Drawing a Coexpressed Gene Network for a Set of Genes
		4 Notes
		References
Index