Lipidomics: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Isolation of Mitochondrial Lipids and Mass Spectrometric Analysis
1 Introduction
2 Materials
2.1 Solvents/Solutions
2.2 Other Materials
2.3 High-Performance Liquid Chromatography and Mass Spectrometry
3 Methods
3.1 Sample Preparation and Mitochondrial Isolation
3.2 Butanol-Methanol (BUME) Lipid Extraction
3.3 High-Performance Liquid Chromatography and Mass Spectrometry (HPLC and MS)
3.3.1 HPLC and MS Methods
3.4 Lipid Identification and Bioinformatics Analysis
4 Notes
References
Chapter 2: Isolation of Neuronal Synaptic Membranes by Sucrose Gradient Centrifugation
1 Introduction
2 Materials
2.1 Solutions
2.2 Equipment
3 Methods
3.1 Isolation Steps
3.2 Wash and Storage Steps
4 Notes
References
Chapter 3: Multi-macromolecular Extraction from Endosymbiotic Anthozoans
1 Introduction
1.1 Safety Considerations
1.2 Drawbacks to Current Approaches
1.3 Justification for a Multi-omic Approach
1.4 Protocol Overview and Rationale
2 Materials
3 Methods
3.1 Sample Collection
3.2 RNA Extraction
3.3 DNA Extraction
3.4 Protein, Lipid, and Metabolite Extractions
3.4.1 Option A (Fig. 5)
Option A, Phase I: Proteins (Fig. 5)
Option A, Phase II: Lipids and Polar Metabolites (Fig. 5)
3.4.2 Option B (Fig. 6)
3.5 Shotgun Proteomics
3.6 Liquid Chromatography/Mass Spectrometry
3.7 iTRAQ
Appendices
References
Chapter 4: High-Resolution Liquid Chromatography-Mass Spectrometry for Lipidomics
1 Introduction
2 Materials
3 Methods
3.1 Sample Preparation
3.2 Mass Spectrometric Analysis
3.2.1 Vanquish Horizon Parameters
3.2.2 HESI Source Parameters
3.2.3 Q Exactive Parameters
3.3 Data Analysis
4 Notes
References
Chapter 5: Characterization of Gangliosides from Mouse Optic Nerve Samples Using Mass Spectrometry
1 Introduction
2 Materials
2.1 Extraction
2.2 High-Performance Liquid Chromatography and Mass Spectrometry
3 Methods
3.1 Gangliosides and Lipid Extraction
3.2 Profiling Using a Q-Exactive Orbitrap Instrument Coupled with Vanquish Horizon Binary UHPLC LC-MS System
3.2.1 LC-Conditions
3.2.2 MS-Conditions
3.2.3 dd-MS2/ dd-SIM
3.3 Identification and Quantification
4 Notes
References
Chapter 6: Protocol and Methods Applicable to Retinal Vascular Diseases
1 Introduction
1.1 Techniques for Measuring the Lipidomics
2 Materials
3 Methods
3.1 Ocular Fluid-Tear and Vitreous Humor Extraction
3.2 Lipid Separation and Identification
3.3 Data Analysis
4 Notes
References
Chapter 7: Analysis and Annotation of Phospholipids by Mass Spectrometry-Based Metabolomics
1 Introduction
2 Materials
2.1 Sample Extraction
2.2 Metabolomics Analysis
3 Methods
3.1 Metabolomics Extraction of Biological Samples
3.1.1 Extraction of Plasma/Serum Samples
3.1.2 Extraction of Erythrocyte Samples
3.1.3 Extraction of Tissue Samples
3.2 Metabolomics Analysis by Direct MS Fingerprinting
3.3 Metabolomics Analysis by UHPLC-MS
3.4 Annotation of Phospholipids in the Positive Ion Mode
3.5 Annotation of Phospholipids in the Negative Ion Mode
4 Notes
References
Chapter 8: Profiling the Mammalian Lipidome by Quantitative Shotgun Lipidomics
1 Introduction
2 Materials
2.1 Sample Preparation
2.2 Mass Spectrometry
3 Methods
3.1 Sample Preparation
3.2 Single-Step Lipid Extraction
3.3 Ionization and Mass Spectrometry
3.4 Data Extraction and Quantification
4 Notes
References
Chapter 9: Lipidomics Analysis with Triple Quadrupole Mass Spectrometry
1 Introduction
2 Materials
2.1 Preparation of Standard and Sample
2.2 Triple Quadrupole Mass Spectrometry
3 Methods
3.1 Preparation of Standard and Sample
3.2 Triple Quadrupole Mass Spectrometry
3.2.1 Analysis of Standard
3.2.2 Sample Analysis
4 Notes
References
Chapter 10: High-Performance Chromatographic Separation of Cerebrosides
1 Introduction
2 Materials
2.1 Special Equipment
2.2 HPTLC
3 Methods
3.1 HPTLC Development Tank Setup
3.2 HPTLC Setup
3.3 Sample Loading
3.4 Chromatogram Development
3.5 Visualization and Quantitative Determination
4 Notes
References
Chapter 11: Quantification of Endocannabinoids from Biological Samples Using Liquid Chromatography-Tandem Mass Spectrometry
1 Introduction
2 Materials
2.1 LC-MS/MS for SPE-Pretreated Tissue Samples
2.1.1 SPE Treatment
2.1.2 LC-MS/MS Analysis for SPE Samples
2.2 LC-MS/MS for In Vivo Brain Microdialysis Samples
2.2.1 Animal Surgery and In Vivo Microdialysis
2.2.2 LC-MS/MS Analysis for Samples from Brain Microdialysis
2.3 Other Supplies
3 Methods
3.1 LC-MS/MS for SPE-Pretreated Tissue Samples
3.1.1 Extraction and Purification of eCBs Using SPE
3.1.2 LC-MS/MS Analysis
3.1.3 Calibration Curves for eCBs Quantification
3.2 LC-MS/MS for In Vivo Brain Microdialysis Samples
3.2.1 Surgery
3.2.2 In Vivo Microdialysis
3.2.3 LC-MS/MS Analysis
4 Notes
References
Chapter 12: Analysis of Lipids, Fatty Acid, and Cholesterol in Membrane Microdomains
1 Introduction
2 Materials
2.1 Solutions for Detergent-Free Preparation of Membrane Microdomains/Caveolae by Carbonate Extraction (Modified from Song et ...
2.2 Solutions for Detergent-Free Preparation of Membrane Microdomains by Simplified Optiprep Method (Macdonald and Pike Method)
2.3 Solutions for Preparation of Detergent-Resistant Membranes (Elliott et al. Modified from Seno et al.)
2.4 Materials for Two-Part Extraction of Saponifiable and Nonsaponifiable Lipids
3 Methods
3.1 Isolation of Detergent-Free Membrane Microdomains/Caveolae by Carbonate Extraction (Modified from Song et al.,)
3.2 Isolation of Detergent-Free Preparation of Membrane Microdomains by Simplified Optiprep Method (Macdonald and Pike Method)
3.3 Isolation Detergent-Resistant Membranes (Elliott et al. Modified from Seno et al.)
3.4 Evaluation of Membrane Domains
3.5 Analytical Approaches to Examine Lipid Composition of Membrane Microdomains
3.5.1 Two-Part Extraction Procedure for Saponifiable and Nonsaponifiable Lipids
3.5.2 Analysis of Cholesterol by High-Performance Liquid Chromatography (HPLC)
3.5.3 Analysis of Membrane Lipid Composition by LC-MS/MS (Modified from Busik et al. and Cheng et al.)
3.5.4 Preparation of Fatty Acid Methyl Esters and Analysis by Gas Chromatography and Flame Ionization Detection
4 Notes
References
Chapter 13: Analysis of Cholesterol Lipids Using Gas Chromatography Mass Spectrometry
1 Introduction
2 Materials
3 Methods
3.1 Extraction of Total Lipids from Tissue Sample
3.2 Gas Chromatography-Mass Spectrometry Analysis
3.2.1 Standard Preparation
3.2.2 Preparation of Lipid Samples
3.2.3 Instrumental Conditions
4 Notes
References
Chapter 14: Analyses and Localization of Phosphatidylcholine and Phosphatidylserine in Murine Ocular Tissue Using Imaging Mass...
1 Introduction
2 Materials
2.1 Solvents
2.2 MALDI/MS Instrument and Accessories
2.3 Software
2.4 Other Materials
2.5 Preparation of Matrix Solutions for Calibration
2.6 Preparation of the Calibration Mix Solutions
2.7 Preparation of Matrix Solution for Tissue
3 Methods
3.1 Tissue Preparation Using Fresh Frozen Tissue Samples
3.2 Matrix Application
3.3 MALDI Source Preparation
3.4 Calibration of Mass Spectrometer
3.5 Data Acquisition from Sample
3.6 Data Analysis
4 Notes
References
Chapter 15: A Robust Phenotypic Screening Assay Utilizing Human Podocytes to Identify Agents that Modulate Lipid Droplets
1 Introduction
2 Materials
2.1 Cells and Cell Culture Reagents
2.2 Equipment
3 Methods
3.1 Coating 96-Well Plates
3.2 Culturing and Plating of Cells
3.3 Fixation of Cells (Done on Day 14)
3.4 Staining
3.5 Image Acquisition Using an Automated Confocal Microscope
3.6 Image Analysis and Quantification of Lipid Droplets
4 Notes
References
Chapter 16: Capillary Electrophoresis Plasma Fractionation for Lipids Analysis
1 Introduction
2 Materials
2.1 Fractionation of Blood
2.2 Capillary Electrophoresis
2.3 Lipid Isolation
3 Methods
3.1 Fractionation of Blood
3.2 CE System Settings
3.3 CE System Priming
3.4 Plasma Fractionation
3.5 Preparation of Samples for Mass Spectrometry Lipidomics
4 Notes
References
Chapter 17: Combined Use of MALDI-TOF Mass Spectrometry and 31P NMR Spectroscopy for the Analysis of (Phospho)Lipids
1 Introduction
1.1 Lipids and Lipid Analysis
1.2 Using the Strengths of MALDI-TOF MS and 31P NMR in the Lipid Field
2 Materials
2.1 Equipment and Supplies
2.1.1 MALDI-TOF MS
2.1.2 31P NMR Spectroscopy
2.1.3 Reagents and Biological Mixtures
3 Methods
3.1 Sample Processing
3.1.1 MALDI-TOF MS of Artificial Lipid Samples of Known Composition
3.1.2 MALDI-TOF MS of the Lipids in the Hen Egg Yolk Extract
3.1.3 Preparation of the 31P NMR Samples
3.2 Recording MALDI-TOF Mass Spectra
3.3 31P NMR Spectroscopy
3.4 Results
4 Notes
References
Chapter 18: Single-Step Capture and Targeted Metabolomics of Alkyl-Quinolones in Outer Membrane Vesicles (OMVs) of Pseudomonas...
1 Introduction
2 Materials
2.1 Reagents and Instrumentation
2.2 Bacterial Cultures and Samples
3 Methods
3.1 Isolation of OMVs from P. Aeruginosa Cultures and Bio-Fluids
3.2 Single-Step Analysis of OMV QS Metabolites by LDI -MS
4 Notes
References
Chapter 19: Isoprenylation of Monomeric GTPases in Human Trabecular Meshwork Cells
1 Introduction
2 Materials
2.1 Primary Human TM Cell Culture
2.2 Transformed Human TM Cell Culture
2.3 Test Reagents
2.4 Electrophoresis and Electrotransfer
2.5 Immunoblotting
3 Methods
3.1 Preparation and Culture of Human Primary TM Cells
3.2 Culture of Human Transformed TM Cells
3.3 Protein Prenylation Alters GTPase Protein Expression
3.4 SDS-PAGE and Electrotransfer
3.5 Immunoblotting
4 Notes
References
Chapter 20: Angiogenesis Model of Cornea to Understand the Role of Sphingosine 1-Phosphate
1 Introduction
2 Materials
2.1 Corneal Wounding
2.2 Cornea Harvest and Immunohistochemistry
3 Methods
3.1 Corneal Wounding
3.2 Cornea Harvest and Immunohistochemistry
3.3 Imaging
3.4 Invasion Percent Quantification
4 Notes
References
Chapter 21: Lipid Extraction and Sample Preservation Techniques for Stable Isotope Analysis and Ecological Assays
1 Introduction
2 Materials
3 Methods
3.1 Subsampling Tissue
3.2 Lipid Extraction by Soxhlet Apparatus
3.3 Lipid Extraction with a Shaker (if a Soxhlet Apparatus Is Not Available, Lipid Extraction Must Be Performed Manually)
3.4 Unpacking and Homogenization
3.5 Weighing
3.6 Processing Hair Samples
4 Notes
References
Chapter 22: Rapid Analysis of Plasmalogen Individual Species by High-Resolution Mass Spectrometry
1 Introduction
2 Materials
2.1 Solution Preparation for Chromatographic Separation
2.2 Solvent Preparation for Lipid Extraction
2.3 Standard Preparation
2.4 Laboratory Glassware and Equipment
3 Methods
3.1 Sample Preparation (Fluids and Biofluids)
3.2 Sample Preparation (Tissues)
3.3 Lipid Extraction
3.4 Liquid Chromatography
3.5 Mass Spectrometry Method
3.6 PlsCho Annotation
4 Notes
References
Chapter 23: Untargeted Analysis of Lipids Containing Very Long Chain Fatty Acids in Retina and Retinal Tight Junctions
1 Introduction
2 Materials
2.1 Reagents and Consumables
2.2 Equipment
3 Methods
3.1 Isolation of Tight Junctions from Retinal Pigment Epithelial Cells
3.2 Lipid Extraction
3.3 Direct Infusion Nano-Electrospray Ionization (nESI) of Tight Junction or Retina Tissue Lipid Extracts
3.4 Analysis of Tight Junction or Retina Lipid Profiles Using Orbitrap High Resolution/Accurate Mass Spectrometry
3.5 Post-Analysis Data Processing and Database Searching to Identify Putative Lipids Containing Very Long Chain Fatty Acids
3.6 Characterization of Lipid Very Long Chain Fatty Acid Constituents by Untargeted Ion Mapping MS/MS
3.7 Processing Untargeted High Resolution/Accurate Mass MS/MS Data to Identify Lipid Species Containing Very Long Chain Fatty ...
4 Notes
References
Chapter 24: Analysis of Lipid Contents in Human Trabecular Meshwork Cells by Multiple Reaction Monitoring (MRM) Profiling Lipi...
1 Introduction
2 Materials
2.1 Dissection of TM from Human Tissue
2.2 Culturing HTM Cells
2.3 Lipid Extraction from HTM Cells
2.4 MRM-Based Lipidomics Analysis
3 Methods
3.1 Dissection of TM from Human Tissue
3.2 Culturing HTM Cells
3.3 Lipid Extraction from HTM Cells (Bligh and Dyer Method)
3.4 MRM-Based Lipidomics Analysis
4 Notes
References
Chapter 25: Mass Spectrometry Approaches for Detection and Determination of Prostaglandins from Biological Samples
1 Introduction
2 Materials
2.1 Solid Tissue/Cell Homogenates´ Preparation
2.2 Solid Phase Extraction
2.3 Off-line LC-MS/MS Analysis
2.4 On-line LC-MS/MS Analysis
2.5 Other Supplies
3 Methods
3.1 Off-line SPE LC-MS/MS
3.1.1 Preparation of Solid Tissue Homogenates Before SPE
3.1.2 Preparation of Cell Homogenates Before SPE
3.1.3 Preparation of Serum/Plasma Samples Before SPE
3.1.4 SPE Procedures
3.1.5 Calibration Curves for PGs Quantification
3.1.6 LC-MS/MS Conditions
3.2 On-Line SPE LC-MS/MS
3.2.1 Tissue Sample Preparation
3.2.2 Calibration Curves for PGs Quantification
3.2.3 LC-MS/MS Conditions
4 Notes
References
Chapter 26: Downloading and Analysis of Metabolomic and Lipidomic Data from Metabolomics Workbench Using MetaboAnalyst 5.0
1 Introduction
2 Materials
2.1 MetaboAnalyst
2.2 Metabolomics Workbench
3 Methods
3.1 Protocol for Downloading Data from Metabolomics Workbench
3.2 Importing and Analyzing Data in MetaboAnalyst 5.0
3.2.1 Statistical Analysis Using MetaboAnalyst 5.0
3.2.2 Targeted and Untargeted Metabolomics Data
4 Notes
References
Chapter 27: Accurate Analysis of Lipid Building Blocks Using the Tool LipidOne
1 Introduction
2 General Information
3 Methods
3.1 Installation
3.2 LipidOne Input Data Format
3.3 Type of Analyses that Can Be Performed by LipidOne
4 Notes
References
Chapter 28: Image-Based Longitudinal Characterization of Corneal Wound to Understand the Role of Sphingosine-1-Phosphate
1 Introduction
2 Materials
2.1 Corneal Alkali Burning
2.2 Corneal Imaging
3 Methods
3.1 Corneal Alkali Burning
3.2 Corneal Imaging
3.3 Image Analysis
4 Notes
References
Chapter 29: Characterization of Trabecular Meshwork Mechanical Property Modulation After Application of Lipids
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Trabecular Meshwork Samples
3.2 Lipid Treatment
3.3 Mechanical Property Measurement Using Microindentation
4 Notes
References
Chapter 30: C-Laurdan: Membrane Order Visualization of HEK293t Cells by Confocal Microscopy
1 Introduction
2 Materials
3 Methods
3.1 HEK293t Cell Sample Staining with C-Laurdan
3.2 Imaging Equipment Setup and Sample Image Acquisition for Leica Stellaris 8 Confocal Microscope
3.3 Calibration Image Acquisition
3.4 Image Analysis by ImageJ
3.4.1 Calculate G Correction Factor
3.4.2 Measure Background Pixel Intensity
3.4.3 Run ImageJ Macro
4 Notes
References
Chapter 31: Sonication-Based Basic Protocol for Liposome Synthesis
1 Introduction
2 Materials
3 Methods
4 Notes
References
Index