Mass Spectrometry-Based Lipidomics: Methods and Protocols

Preface
	Reference
Contents
Contributors
Chapter 1: Mass Spectrometry-Based Lipidomics: An Overview
	1 Introduction
	2 Lipidomics and Chromatographic Separation
		2.1 Lipid Extraction
		2.2 Chromatographic Separation
	3 Mass Spectrometric Analysis and Bioinformatics
	4 Conclusion and Perspective
	References
Chapter 2: High Confidence Shotgun Lipidomics Using Structurally Selective Ion Mobility-Mass Spectrometry
	1 Introduction
		1.1 Challenges in Lipidomic Analyses
		1.2 Ion Mobility of Lipids
		1.3 Instrumentation and Software Overview
	2 Materials
		2.1 Sample Preparation
		2.2 Solutions
		2.3 Data Acquisition
		2.4 Data Preprocessing
		2.5 Feature Extraction and Annotation
	3 Methods
		3.1 Sample Preparation
		3.2 Instrument Tuning and Calibration
		3.3 Data Acquisition Methods Preparation
			3.3.1 CCS Calibration Methods Preparation
			3.3.2 Shotgun Lipidomics IM-MS Methods Preparation
		3.4 IM-MS Data Acquisition
		3.5 Data Preprocessing Using PNNL PreProcessor
			3.5.1 Drift Time Smoothing Using the PNNL PreProcessor
			3.5.2 Single-Field CCS Calibration Using IM-MS Browser
			3.5.3 IM-MS Data Filtering Using IM-MS Browser
		3.6 Feature Extraction Using MassHunter Mass Profiler
		3.7 Annotation of Lipidomic Features Using CCS
			3.7.1 Molecular Annotation Using Database Searching in MassHunter ID Browser
		3.8 Export Features and Prepare a Targeted MS/MS  List
		3.9 Targeted Fragmentation Methods Preparation
		3.10 Targeted Fragmentation Qualitative Data Analysis
	4 Notes
	References
Chapter 3: Global Lipidomics Profiling by a High Resolution LC-MS Platform
	1 Introduction
	2 Materials
		2.1 Chemicals and Equipments
		2.2 Lipid Extraction
		2.3 Lipid Standards
		2.4 HPLC/MS
	3 Methods
		3.1 Preparation of Standard Mixes
		3.2 Lipid Extraction
		3.3 Sample Preparation for Positive Ion Electrospray Ionization (ESI)
		3.4 Sample Preparation for Negative Ion ESI Analysis
		3.5 Instrument Settings for LC/MS Analysis
			3.5.1 UHPLC
			3.5.2 Mass Spectrometry
		3.6 Data Acquisition
		3.7 Data Processing
		3.8 Data Analysis
	4 Notes
	References
Chapter 4: Comprehensive Structural Characterization of Lipids by Coupling Paternò-Büchi Reaction and Tandem Mass Spectrometry
	1 Introduction
	2 Materials
	3 Methods
		3.1 Cell Sample Preparation
		3.2 Tissue Sample Preparation
		3.3 Lipid Extraction
		3.4 PB Reaction and MS Analysis
		3.5 PB Derivatization Using 2-Acetylpyridine for Determining Both C=C Locations and sn-Position of Fatty Acyls in Phospholipids
	4 Notes
	References
Chapter 5: Chemical Derivatization-Aided High Resolution Mass Spectrometry for Shotgun Lipidome Analysis
	1 Introduction
	2 Materials
		2.1 Lipid Extraction
		2.2 Lipid Derivatization
		2.3 Consumables and Equipment
	3 Methods
		3.1 Monophasic Lipid Extraction
		3.2 Functional Group Selective Lipid Derivatization Reactions
			3.2.1 Functional Group Selective Derivatization of Aminophospholipids
			3.2.2 Functional Group Selective Derivatization of Plasmalogen-Containing Lipids
	4 Notes
	References
Chapter 6: Quantitative Analysis of Polyphosphoinositide, Bis(monoacylglycero)phosphate, and Phosphatidylglycerol Species by S...
	1 Introduction
	2 Materials
		2.1 Tissue Harvest and Homogenization
		2.2 Shotgun Lipidomics
	3 Methods
		3.1 Tissue Harvest and Homogenization
		3.2 Shotgun Lipidomics for Accurate, Comprehensive, and High-Throughput Analysis of Isomeric PG and BMP
			3.2.1 Lipid Extraction for PG and BMP Analysis
			3.2.2 Permethylation of PG with TMS-D Reagent
			3.2.3 Mass Spectrometric Analysis of Methylated PG and BMP Species
		3.3 Shotgun Lipidomics for Accurate, Comprehensive Analysis of PIP, PIP2, and PIP3
			3.3.1 Lipid Extraction
				Lipid Extraction with Modified MTBE Extraction Method
				Lipid Extraction with Modified Folch Extraction Method
			3.3.2 Permethylation of PPI with TMS-D Reagent
			3.3.3 Mass Spectrometric Analysis of Methylated PPI Species
	4 Notes
	References
Chapter 7: Mass Spectrometry-Based Shotgun Lipidomics Using Charge-Switch Derivatization for Analysis of Complex Long-Chain Fa...
	1 Introduction
	2 Materials
		2.1 Alkaline or Acid Hydrolysis to Release Free Fatty Acids
		2.2 Reagents for Preparation of FA-AMPP Derivatives
		2.3 Sample Preparation for MALDI TOF/TOF Analysis
		2.4 High-Resolution Tandem Mass Spectrometric Analysis
	3 Methods
		3.1 Preparation of Hydroxyphthioceranoic and Phthioceranoic Acids
		3.2 Preparation of Other Microbial Long-Chain Fatty Acids
		3.3 Preparation of FA-AMPP Derivative
		3.4 MALDI-TOF/TOF Structural Analysis of FA-AMPP Derivatives
		3.5 ESI Tandem Mass Spectrometric Analysis of the FA-AMPP Derivatives as M+  Ions
	4 Notes
	References
Chapter 8: One-Pot Extractive Transesterification of Fatty Acids Followed by DMOX Derivatization for Location of Double Bonds ...
	1 Introduction
	2 Materials and Equipment
		2.1 Preparation of Biological Samples
		2.2 Transesterification and Extraction of FAMEs
		2.3 Formation of DMOX Derivative
		2.4 GC/MS Analysis
	3 Methods
		3.1 Preparation of Biological Samples
			3.1.1 Cell Culture
			3.1.2 Plant Tissue
		3.2 Extractive Transesterification to Generate Fatty Acid Methyl Esters (FAMEs) (Fig. 1)
		3.3 Dimethyloxazoline (DMOX) Derivatives from FAMEs (Fig. 2) [3, 18, 20, 21  ]
		3.4 GC/MS Analysis
		3.5 Data Analysis
			3.5.1 Evaluation of Chromatography
			3.5.2 Generating Suitable Mass Spectra and Assigning Identification
			3.5.3 Quantification
	4 Notes
	References
Chapter 9: Ceramide Analysis by Multiple Linked-Scan Mass Spectrometry Using a Tandem Quadrupole Instrument
	1 Introduction
	2 Materials
		2.1 Ceramide Preparation and Extraction
		2.2 Other Crude Ceramide Mixtures
		2.3 Mass Spectrometric Analysis
	3 Methods
		3.1 Extraction of Ceramide from Dandruff Flakes
		3.2 Preparation of Crude Ceramide for Linked-Scan Data Acquisition
		3.3 Mass Spectrometric Analysis Using Linked Scannings with a TSQ Instrument
	4 Notes
	References
Chapter 10: Comprehensive Mouse Skin Ceramide Analysis on a Solid-Phase and TLC Separation with High-Resolution Mass Spectrome...
	1 Introduction
	2 Materials
		2.1 Collection of Newborn Mouse Epidermis
		2.2 Extraction of Non-bound (Free, Extractable) Lipids
		2.3 Extraction of Protein-Bound Lipids
		2.4 TLC Separation
		2.5 Preparative  TLC
		2.6 Fractionation of Lipids by SPE Columns
		2.7 High-Resolution Mass Spectrometric Analysis
	3 Methods
		3.1 Collection of Mouse Newborn Epidermis
		3.2 Extraction of Non-bound (Free, Extractable) Lipids
		3.3 Extraction of Protein-Bound Lipids
		3.4 TLC Separation
		3.5 Preparative  TLC
		3.6 Fractionation of Lipids by SPE Columns
		3.7 High-Resolution Mass Spectrometric Analysis
	4 Notes
	References
Chapter 11: Mass Spectrometric Analysis of Meibomian Gland Lipids
	1 Introduction
	2 Materials
	3 Methods
		3.1 Meibum Sample Collection
		3.2 Sample Solution Preparation
		3.3 MS and MS/MS Data Acquisition
	4 Notes
	References
Chapter 12: Quantification of Plasma Oxylipins Using Solid-Phase Extraction and Reversed-Phase Liquid Chromatography-Triple Qu...
	1 Introduction
	2 Materials
		2.1 Instruments and Consumables
		2.2 Standard Stock Solution and Mixture
		2.3 Solvents for SPE (for 1 Plate of 96 Samples)
		2.4 Solvents for LC-MRM-MS
	3 Methods
		3.1 SPE Cleanup
		3.2 Setting Up MRM Transitions
		3.3 LC-MRM-MS Analysis
		3.4 Quality Control and Data Analysis
	4 Notes
	References
Chapter 13: Targeted Lipidomics of Drosophila melanogaster During Development
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Reagents and Solvents
			2.2.1 Lipid Extraction
			2.2.2 Mass Spectrometry
				Internal Standards
				Mobile Phase Solvents (See Note 1)
				LC Columns (See Note 1)
		2.3 Animals
		2.4 Supplies
	3 Methods
		3.1 Lipids Extraction Procedure
		3.2 Sample Preparation for Mass Spectrometry Analysis
		3.3 Preparation of LC
			3.3.1 Preparation of Mobile Phases for Normal Phase LC
			3.3.2 Preparation of 0.1 M Ammonium Acetate Stock Solution
			3.3.3 Preparation of Mobile Phases for Reversed Phase LC
			3.3.4 LC Conditions
				Normal Phase LC
				Reversed Phase LC
		3.4 MS Instrumentation
			3.4.1 ESI-MS
			3.4.2 APCI-MS
		3.5 Semi-Quantitation of D. melanogaster Lipids Using MRM and SIM
		3.6 Data Analysis
	4 Lipid Found in D. melanogaster at Different Stages of Its Life Cycle
		4.1 Sphingolipids (SPL)
		4.2 Glycerophospholipids (GPL)
		4.3 Sterol Ester
		4.4 Triacylglycerols (TAG)
	5 Notes
	References
Chapter 14: Shotgun Lipidomic Analysis of Leishmania Cells
	1 Introduction
	2 Materials
		2.1 Media for Leishmania Parasites (See Note 1)
		2.2 Lipid Extraction
		2.3 High-Resolution Mass Spectrometric Analysis
		2.4 Tandem Quadrupole Linked Scan Mass Spectrometric Analysis
	3 Methods
		3.1 Cultivation of Leishmania Promastigotes or Axenic Amastigotes (See Note 5)
		3.2 Preparation of Total Leishmanial Lipids by the Bligh and Dyer Method (See Note 7)
		3.3 Preparation of Total Leishmanial Lipids by the Folch Method (See Note 11)
		3.4 High-Resolution ESI/MSn Analysis Using a LTQ Orbitrap
		3.5 ESI Tandem Quadrupole MS/MS Analysis by Thermo TSQ Vantage EMR (See Note 13)
		3.6 Data Processing for High-Resolution Mass Spectra
	4 Notes
	References
Chapter 15: Characterization of the Uncommon Lipid Families in Corynebacterium glutamicum by Mass Spectrometry
	Abbreviations
	1 Introduction
	2 Materials
		2.1 Complex Medium,  BHI
		2.2 Lipid Extraction
		2.3 Preparative HPLC
		2.4 High-Resolution Mass Spectrometric Analysis
	3 Methods
		3.1 Preparation of Complex Medium, BHI (Follows the Procedures Described by Eggeling and Bott)
		3.2  Bacterial Culture
		3.3 Lipid Extraction
			3.3.1 Apolar Lipids
			3.3.2 Polar Lipids (See Note 11)
		3.4 Preparative HPLC
		3.5 Mass Spectrometric Analysis
	4 Notes
	References
Chapter 16: Mass Spectrometric Analysis of Bioactive Sphingolipids in Fungi
	1 Introduction
	2 Materials
		2.1 Fungal Strains
		2.2 Cell Culture
		2.3 Chemicals
		2.4 Equipments
	3 Methods
		3.1 Culture Collections
		3.2 Lipid Extraction
			3.2.1 Mandala Extraction [27]
			3.2.2 Post Mandala Extraction
		3.3 Estimation of Phosphate (Pi) Content
		3.4 HPLC Separations
		3.5 MS Analysis
		3.6 Data Analysis
			3.6.1 Lipid Quantification
			3.6.2 Data Normalization
	4 Notes
	References
Chapter 17: Analytical Methodologies for Lipidomics in Hemp Plant
	1 Introduction
	2 Materials
		2.1 Plant Material Preparation for UHPLC-HRMS Analysis
		2.2 Equipment, Instrumentation, and Software
		2.3 Solvent Mix for SPE Separation
	3 Methods
		3.1 Lipid Extraction
		3.2 GCB-SPE Purification
		3.3 LC-MS Analysis (See Note 5)
			3.3.1 Set Up the UHPLC System as Follows
			3.3.2 Set Up the Mass Spectrometry as Follows (Unit Is Arbitrary Unless Specified) (See Note 6)
		3.4 Data Analysis by the Lipostar Software (Molecular Discovery, Borehamwood, UK)
			3.4.1 Activate ``LC-MS Settings´´  Page
			3.4.2 Activate ``Lipid Identification Methods´´  Page
			3.4.3 Activate ``Sample Properties´´  Page
			3.4.4 Activate ``Lipostar Preferences´´  Page
		3.5 Data Processing
		3.6 Data Analysis
	4 Notes
	References
Chapter 18: Shotgun Bacterial Lipid A Analysis Using Routine MALDI-TOF Mass Spectrometry
	1 Introduction
	2 Materials
		2.1 Bacterial Culture
		2.2 Sample Preparation for MALDI  MS
		2.3 MALDI MS Analysis
	3 Methods
		3.1 Growth of Bacteria
		3.2 Harvest and Lysis of Bacteria
		3.3 Matrix Preparation
		3.4 Loading the Samples into the MALDI Target Plate
		3.5 MALDI Acquisition
	4 Notes
	References
Chapter 19: Imaging of Polar and Nonpolar Lipids Using Desorption Electrospray Ionization/Post-photoionization Mass Spectromet...
	1 Introduction
	2 Materials
		2.1 Tissue Preparation
		2.2 DESI/PI Source
		2.3 Instrumentation
		2.4 Software
	3 Methods
		3.1 Preparation of Tissue Samples
		3.2 DESI/PI
		3.3 DESI/PI Signal Optimization
		3.4 Imaging Protocols
		3.5 Results
	4 Notes
	References
Chapter 20: Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging of Lipids in the Ischemic Rat Brain Section:...
	1 Introduction
	2 Materials
		2.1 Chemicals, Solutions, and Consumables
		2.2 Equipment
		2.3 Miscellaneous Items
	3 Methods
		3.1 Rat Brain Collection and Handling
		3.2 Cryosection and Tissue Section Processing
		3.3 MALDI Matrix Application
		3.4 MSI Data Acquisition
		3.5 Data Analysis
	4 Notes
	References
Index