Clinical Metabolomics: Methods and Protocols

Preface
Contents
Contributors
Part I: Clinical Metabolomics
	Chapter 1: (Pre)Clinical Metabolomics Analysis
		1 Background and Definitions
		Box 1: Metabolomics Definitions
		2 Metabolomics in Biological and Clinical Context
		3 Analytical Technologies
			3.1 Gas Chromatography-Mass Spectrometry (GC-MS)
			3.2 Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
			3.3 Nuclear Magnetic Resonance Spectroscopy (NMR)
			3.4 Capillary Electrophoresis Mass Spectrometry (CE-MS)
			3.5 Upcoming Technologies
		4 Bioinformatics
		5 Concluding Remarks
		References
Part II: LC-MS-Based Metabolomics
	Chapter 2: Global Metabolomics Using LC-MS for Clinical Applications
		1 Introduction
		2 Materials
			2.1 Samples
			2.2 Chemicals and Standards
			2.3 Solutions and Solvents
			2.4 LC-MS Instrumentation
			2.5 Software
			2.6 Other Equipment and Consumables
		3 Methods
			3.1 Sample Preparation
				3.1.1 Preparation of Plasma and Serum Samples
				3.1.2 Preparation of Dried Blood Spot Samples
				3.1.3 Preparation of Urine Samples
				3.1.4 Preparation of Cerebrospinal Fluid Samples
			3.2 Liquid Chromatographic Settings
			3.3 Electrospray Ionization Settings
			3.4 Mass Spectrometric Settings
			3.5 Analysis Sequence Setup
			3.6 Data Processing and Workflow
			3.7 Evaluation of Data Quality Prior to Interpretation
			3.8 Metabolite Identification and Interpretation
		4 Notes
		References
	Chapter 3: Modular, Scalable, and Customizable LC-HRMS for Exposomics
		1 Introduction
		2 Materials
			2.1 System Suitability  Test
			2.2 Analytical Standards
			2.3 Isotopically Labeled Internal Standards
			2.4 Biological Samples
			2.5 Sample Extraction
			2.6 Liquid Chromatography-Mass Spectrometry
		3 Methods
			3.1 System Suitability
			3.2 Sample Extraction
			3.3 Liquid Chromatography-Mass Spectrometry
		4 Data Analysis
			4.1 Non-targeted Analysis (NTA) and Suspect Screening Analysis (SSA)
			4.2 Semi-targeted Data Analysis
		5 Notes
		References
	Chapter 4: Distinguishing Artifactual Fatty Acid Dimers from Fatty Acid Esters of Hydroxy Fatty Acids in Untargeted LC-MS Pipe...
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Solvents
			2.3 Software and Related Files
		3 Methods
			3.1 Extraction of Polar and Nonpolar Metabolites from Serum for LC-MS-Based Metabolomics
			3.2 Liquid Chromatography-Mass Spectrometry Analysis and DDA of Serum Samples
				3.2.1 DDA for In-Depth Enhanced Tandem MS Database Analysis
			3.3 Data Analysis
				3.3.1 Data Processing
				3.3.2 Differential Analysis
			3.4 Identification of Putative Metabolites and Removal of Artifactual FA Dimers from Untargeted Results
		4 Notes
		References
	Chapter 5: Advances in Chiral Metabolomic Profiling and Biomarker Discovery
		1 Introduction
		2 Physiological Functions and Metabolism of D-Amino Acids and D-Hydroxy Acids in Mammals
		3 Chromatographic and Mass Spectrometry Techniques for Enantioselective Detection
		4 Novel Biological Insights Through Chiral Metabolomics in Disease Pathophysiology
			4.1 Neurodegenerative Diseases
			4.2 Chronic Kidney Disease
			4.3 Cancer
			4.4 Organic Acidemias and Associated Diseases
		5 Chiral Metabolomics in Clinical Diagnostics: Challenges and Future Prospects
		References
	Chapter 6: Stable Isotope Tracing Experiments Using LC-MS
		1 Introduction
		2 Materials
			2.1 Cell Culture and Isotope Tracing
			2.2 Metabolite Extraction and Sample Preparation
			2.3 LC-MS Analysis
		3 Methods
			3.1 Cell Culture
			3.2 Stable Isotope Tracing
			3.3 Metabolite Extraction
			3.4 Sample Preparation for LC-MS Analysis
			3.5 LC-MS Analysis
			3.6 Data Analysis
		4 Notes
		References
	Chapter 7: Quantification of Total Ketone Bodies in Biological Matrices Using Stable Isotope-Labeled Internal Standards and RP...
		1 Introduction
		2 Materials
			2.1 General Materials and Equipment
			2.2 Materials for Synthesis of [U-13C4] AcAc Internal Standard (Subheading 3.1)
			2.3 Materials for Quantification of Newly Synthesized [U-13C4] AcAc (Subheading 3.2)
			2.4 Materials for Formal Quantification of Endogenous Ketone Bodies in Biological Matrices (Subheading 3.3)
			2.5 Materials for D- and L-βOHB Enantiomeric Separation (Subheading 3.4)
		3 Methods
			3.1 Synthesis of an Authentic [U-13C4] AcAc Stable Isotope-Labeled Internal Standard
			3.2 Quantification of Newly Synthesized [U-13C4]  AcAc
			3.3 Quantifying Ketone Bodies in Multiple Biological Matrices (See Notes 7 and 8)
				3.3.1 Sample Preparation for Measuring Ketone Bodies in Serum or Cell Culture Media
				3.3.2 Sample Preparation for Measuring Ketones in Tissues or Cultured Cells (See Note 14)
				3.3.3 RP-UHPLC-MS/MS Analysis of Ketone Bodies
			3.4 Analytical Separation and Quantification of D- and L-βOHB Enantiomers
		4 Notes
		References
	Chapter 8: LC-MS/MS Quantification of Endocannabinoids in Tissues
		1 Introduction
		2 Materials
			2.1 Samples
			2.2 Standard Solutions
			2.3 Liquid Chromatography-Tandem Mass Spectrometry
		3 Methods
			3.1 Tissue Homogenization
			3.2 Extraction of ECBs
				3.2.1 Extraction of ECBs for Brain, Muscle, Liver, and WAT Tissue
				3.2.2 Extraction of ECBs for BAT Tissue
			3.3 Extract Reconstitution
			3.4 Calibration Line Preparation
			3.5 LC-MS/MS Analysis
			3.6 Data Processing
		4 Notes
		References
	Chapter 9: Prostaglandin Metabolites Analysis in Urine by LC-MS/MS
		1 Introduction
		2 Materials
		3 Methods
			3.1 Preparation of Calibrator and Internal Standard solutions
			3.2 Sample Preparation of Urine
			3.3 Analysis by Liquid Chromatography
			3.4 Analysis by Tandem Mass Spectrometry
			3.5 Data Analysis
		4 Notes
		References
	Chapter 10: A Method for Analysis of Oxidized Phospholipids from Biological Samples Using Mass Spectrometry
		1 Introduction
		2 Materials
			2.1 Equipment for oxPLs Generation and Purification
			2.2 Reagents and Solutions for OxPLs Generation and Purification
			2.3 Equipment for LC-MS/MS Analysis of oxPLs
			2.4 Reagents and Solutions for LC-MS/MS Analysis of oxPLs
		3 Methods
			3.1 Air Oxidation of PL Standards
			3.2 Purification of oxPL Isomers
			3.3 Saponification of oxPLs for Quantification of Individual Positional Isomers
			3.4 Generation of KETE-PL Standards
			3.5 Generation of a Standard Curve
			3.6 Sample Preparation
			3.7 LC-MS/MS Analysis of oxPLs
			3.8 Data Analysis
			3.9 Detection of Partially Characterized eoxPL in Cells and Tissue Samples
		4 Notes
		References
	Chapter 11: Nonenzymatic Oxidized Polyunsaturated Fatty Acid Products in Human Plasma and Urine Samples by LC-QTOF-MS/MS
		1 Introduction
		2 Materials
			2.1 PUFA Oxidation Product Extraction
			2.2 Solid Phase Extraction (SPE)
			2.3 LC-ESI-QTOF-MS/MS Analysis
		3 Methods
			3.1 PUFA Oxidation Product Extraction from Plasma and Urine Samples
				3.1.1 Plasma Samples (See Note 1)
				3.1.2 Urine Samples
			3.2 Solid Phase Extraction (SPE)
			3.3 LC-ESI-QTOF-MS/MS Analysis
		4 Notes
		References
	Chapter 12: Reversed-Phase Ultrahigh-Performance Liquid Chromatography-Mass Spectrometry Method for High-Throughput Lipidomic ...
		1 Introduction
		2 Materials
			2.1 Reagents and Chemicals
			2.2 Lipid Standards
			2.3 Instruments
			2.4 Solvents for RP-UHPLC/MS Analysis
		3 Methods
			3.1 Sample Preparation
			3.2 RP-UHPLC/MS Conditions
			3.3 Data Processing
		4 Notes
		References
	Chapter 13: High-Throughput RPLC-MS/MS Quantification of Short- and Medium-Chain Fatty Acids
		1 Introduction
		2 Materials
			2.1 Standards
			2.2 Reagents
			2.3 Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS)
			2.4 Additional Equipment
		3 Methods
			3.1 Sample Preparation
			3.2 UHPLC-MS/MS Analysis
		4 Notes
		References
	Chapter 14: Long Chain Base Profiling with Multiple Reaction Monitoring Mass Spectrometry
		1 Introduction
		2 Materials
			2.1 Liquid Chromatography-Mass Spectrometry
			2.2 Lipid Extraction Solvent and Alkaline Water
		3 Methods
			3.1 Sample Preparation, Extraction, and Hydrolysis
			3.2 Liquid Chromatography-Mass Spectrometry
			3.3 Data Analysis
		4 Notes
		References
	Chapter 15: Sphingolipid Analysis in Clinical Research
		1 Introduction
		2 Materials
			2.1 Blood Collection
			2.2 Sample Extraction and Derivatization
			2.3 Liquid Chromatography Mass Spectrometry Systems
			2.4 Reagents and Solutions
				2.4.1 Internal Standards (ISTDs)
				2.4.2 Processing and Lipid Extraction
				2.4.3 Mobile Phases for Sphingolipid LC-MS Analysis
				2.4.4 Mobile Phases for SPBP LC-MS Analysis
		3 Methods
			3.1 Experimental Study Design
			3.2 Blood Collection and Preparation of Plasma and Serum
			3.3 Preparation of Plasma/Serum and BQC (Batch Quality Control) Samples
			3.4 Lipid Extraction
			3.5 Derivatization
			3.6 Liquid Chromatography Mass Spectrometry Analysis
				3.6.1 LC-MS Setup for Sphingolipids Analysis
				3.6.2 LC-MS Setup for SPBP Analysis
				3.6.3 LC-MS Analyses
			3.7 Data Analysis
				3.7.1 Peak Integration of MRM Chromatograms
				3.7.2 Normalization and Quantification
				3.7.3 Quality Control
		4 Notes
		References
	Chapter 16: Recommendations for Accurate Lipid Annotation and Semi-absolute Quantification from LC-MS/MS Datasets
		1 Introduction
		2 Material and Equipment Guidelines
			2.1 Reagents and Consumables
			2.2 Lipid Standards
			2.3 Liquid Chromatography: Mass Spectrometry System
			2.4 Data Processing
		3 Methods
			3.1 Deep Lipidomics Profiling and Accurate Annotation of Lipid Molecular Species in a Lipidome of Interest
			3.2 Selection of ISTDs and Design of Lipidome-Specific ISTD Mixture
			3.3 Batch Extraction and Data Acquisition for Semi-absolute Quantification of Individual Samples
			3.4 Data Processing for Semi-absolute Lipid Quantification
		4 Notes
		References
Part III: GC-MS-Based Metabolomics
	Chapter 17: Rapid Analysis of Sterols in Blood-Derived Samples
		1 Introduction
		2 Materials
			2.1 Sample Preparation
			2.2 Materials for GC-MS
		3 Methods
			3.1 Cholesterol Calibration
			3.2 Precursors and Hydroxylated Sterols Calibration
			3.3 Sample Preparation
			3.4 Solid Phase Extraction
			3.5 GC-MS Analysis
			3.6 Data Processing
		4 Notes
		References
Part IV: SFC-MS-Based Metabolomics
	Chapter 18: Ultrahigh-Performance Supercritical Fluid Chromatography-Mass Spectrometry in the Clinical Lipidomic Analysis
		1 Introduction
		2 Materials
			2.1 Reagents, Solvents, and Solutions
			2.2 Lipid Standards
			2.3 Instruments
			2.4 Solvents for UHPSFC/MS Analysis
		3 Methods
			3.1 Sample Preparation
			3.2 UHPSFC/MS Conditions
			3.3 Data Processing
		4 Notes
		References
	Chapter 19: Quantification of Octadecanoids in Human Plasma Using Chiral Supercritical Fluid Chromatography-Tandem Mass Spectr...
		1 Introduction
		2 Materials
			2.1 Working Solutions and Calibration Curve
			2.2 Other Reagents and Materials
			2.3 Solid-Phase Extraction (SPE)
			2.4 Supercritical Fluid Chromatography-Tandem Mass Spectrometry System (SFC-MS/MS)
		3 Methods
			3.1 Trefoil AMY1 Chiral Column Specific Conditioning
			3.2 Sample Preparation
			3.3 Supercritical Fluid Chromatography-Tandem Mass Spectrometry (SFC-MS/MS)
			3.4 SFC-MS/MS Run and Batch Design
			3.5 Data Analysis and Quantification
		4 Notes
		References
	Chapter 20: Rapid Lipid Mediator Profiling by Convergence Chromatography-MS/MS
		1 Introduction
		2 Materials
		3 Methods
			3.1 Homogenization of Tissues
			3.2 Calibrator and Internal Standard Preparation
			3.3 Sample Preparation of Plasma and Tissue Homogenate
			3.4 Analysis by UPC2
			3.5 Mass Spectrometric Detection
			3.6 Data Analysis
		4 Notes
		References
Part V: FIA-MS-Based Metabolomics
	Chapter 21: Analysis of the Mammalian Lipidome by DMS Shotgun Lipidomics
		1 Introduction
		2 Materials
			2.1 Solutions and Solvents
			2.2 Standard Preparation
			2.3 Tuning and Suitability Test Mixes
			2.4 Standard Addition/Sample Extraction
				2.4.1 Sample Preparation-Shimadzu LC-30/Sciex 5500
				2.4.2 Sample Preparation-Shimadzu LC-40/Sciex 6500+
				2.4.3 LC-MS System-Shimadzu LC-30/Sciex 5500
				2.4.4 LC-MS System-Shimadzu LC-40/Sciex 6500+
			2.5 Software
		3 Method
			3.1 Tuning Run
				3.1.1 Tuning Run-Shimadzu LC-30/Sciex 5500
				3.1.2 Tuning Run-Shimadzu LC-40/Sciex 6500+
			3.2 Tuning Analysis
			3.3 Suitability Test
			3.4 Sample Run
			3.5 Data Analysis
		4 Notes
		References
	Chapter 22: Differential Mobility Spectrometry-Based Cardiolipin Analysis
		1 Introduction
		2 Materials
			2.1 Tissue Collection and Mitochondria Isolation
			2.2 Standards and Solutions
			2.3 Material for Tissue Homogenization and Liquid/Liquid Extraction
			2.4 DMS-Based Shotgun Lipidomics
		3 Methods
			3.1 Liver Homogenization Prior to Liquid/Liquid Extraction
			3.2 Liquid/Liquid Extraction of Mitochondria/Liver Homogenate (See Note 7)
			3.3 MS Parameters
			3.4 LC Parameters
			3.5 Tuning of the DMS  Cell
			3.6 System Suitability Test (SST)
			3.7 Sample Analysis and Quantification
		4 Notes
		References
Part VI: CE-MS-Based Metabolomics
	Chapter 23: CE-MS-Based Clinical Metabolomics of Human Plasma
		1 Introduction
		2 Materials
			2.1 Standard and Reference Solutions
			2.2 CE BGE and Sheath Liquid Solutions
			2.3 Human Plasma Collection
			2.4 Capillary Electrophoresis-Mass Spectrometry Instrumentation
			2.5 Other Equipment
			2.6 Software and Databases
		3 Methods
			3.1 Metabolite Extraction, Quality Control Samples, and Biological Sample Preparation
			3.2 Capillary Electrophoresis-Mass Spectrometry Instrumentation
				3.2.1 Cation Analysis
				3.2.2 Anion Analysis
			3.3 Data Preprocessing and Data Analysis
				3.3.1 Data Import and Inspection
				3.3.2 Run Alignment
				3.3.3 Peak Picking, Peak Deconvolution, and Integration
				3.3.4 Data Preprocessing
				3.3.5 Multivariate and Univariate Analysis
				3.3.6 Feature Annotation and Identification
				3.3.7 Pathway Analysis
		4 Case Study
			4.1 Materials and Methods
				4.1.1 Study Design and Classification Criteria
				4.1.2 Data Treatment, Data Analyses, and Feature Annotation
			4.2 Results
				4.2.1 Metabolic Profiling
				4.2.2 Data Analysis
				4.2.3 Metabolite Identification and Biological Interpretation
		5 Notes
		References
Part VII: NMR-Based Metabolomics
	Chapter 24: Quantitative Blood Serum IVDr NMR Spectroscopy in Clinical Metabolomics of Cancer, Neurodegeneration, and Internal...
		1 Introduction
			1.1 Quantitative NMR Spectroscopy and Clinical Metabolomics
			1.2 IVDr NMR Spectroscopy of Serum Metabolites and Lipoproteins
			1.3 IVDr NMR-Based Tools for Quality Control
			1.4 Major Confounders That Impact Serum IVDr NMR  Data
		2 Application of Blood Serum IVDr NMR to Cancer
			2.1 IVDr NMR to Study Blood Serum of Solid Cancers
			2.2 IVDr Serum NMR to Stratify Ovarian Cancer
		3 Application of Blood Serum IVDr NMR to Neurodegeneration
			3.1 Quantitative Serum NMR to Study Neurodegeneration
			3.2 Blood Serum IVDr NMR to Stratify Dementia
		4 Application of Blood Serum IVDr NMR to Internal Medicine
			4.1 Blood Serum IVDr NMR in SARS-CoV-2 Infection
			4.2 Hypertension as Major Confounder in SARS-CoV-2 Infection
		5 Future Perspectives
		References
	Chapter 25: Lipoprotein (Sub)Fraction Analysis on the Bruker B.I. LISA Platform
		1 Introduction
		2 Materials
			2.1 Instrumentation
			2.2 Consumables, Samples, and Buffers
		3 Methods
			3.1 Sample Temperature
			3.2 Sucrose Validation
				3.2.1 Basic Shim Values, Pulse Length P1, and Carrier Frequency Offset O1
				3.2.2 Water-Suppression Performance Test
				3.2.3 Recording the Gradient Profile and Gradient Performance Reference Data
			3.3 QuantRefC Calibration/Validation
			3.4 EDTA Plasma/Serum Sample Preparation
			3.5 NMR Measurements
			3.6 Data Collection
		4 Notes
		References
	Chapter 26: NMR-Based Stable Isotope Tracing of Cancer Metabolism
		1 Introduction
		2 Materials
			2.1 General Considerations
			2.2 Processing Software
		3 Methods
			3.1 Preparation of Extracts for High-Resolution NMR
			3.2 Analysis of Macromolecules
				3.2.1 Protein Digestion for NMR Analysis
				3.2.2 RNA Digestion for NMR Analysis
				3.2.3 Glycogen Digestion for NMR Analysis
			3.3 NMR Analysis of Polar Extracts
				3.3.1 Common NMR Experiment Setup and Processing
					1D PRESAT Procedure (Proton with Water Presaturation)
					1D HSQC Procedure
					2D HSQC Procedure
					1H-1H 2D TOCSY Spectra
					Spectral Deconvolution in MNOVA
				3.3.2 Factors to Consider for Absolute Quantification and Normalization
			3.4 Identification of Isotopomers
			3.5 Multiplex Labeling and Isotope Selection
			3.6 Quantification of Isotopomers: Relative Enrichment from 1D NMR
			3.7 Analysis of Lipid Extracts
			3.8 Corrections for Differential Relaxation
			3.9 Flux Analysis and Kinetics
		4 Notes
		References
	Chapter 27: NMR-Based Analysis of Cellular Central Energy Metabolism and Exchange Rates
		1 Introduction
		2 Materials
			2.1 Collection and Extraction of Culture Media
			2.2 Quenching Intracellular Metabolism of Adherent Cells
			2.3 Extraction of Cells
			2.4 NMR Spectroscopy
			2.5 Software for Analysis
		3 Methods
			3.1 Cell Culture Medium Sample
			3.2 Quenching of Cellular Metabolism
			3.3 Extraction of Intracellular Metabolites
			3.4 Preparation of NMR Samples
			3.5 Preparation of NMR Reference Samples
			3.6 NMR Spectroscopy
			3.7 Quantification of Internal Standard
			3.8 Identification and Quantification of Metabolites
			3.9 Calculation of Exchange Flux Rates
		4 Notes
		References
Part VIII: MALDI-MS-Based Metabolomics
	Chapter 28: In Situ Isotope Tracing at Single-Cell Resolution Using Mass Spectrometry Imaging
		1 Introduction
		2 Materials
			2.1 Tissue Collection
			2.2 Vibratome Sectioning
			2.3 Incubation with Isotope-Labeled Nutrients
			2.4 MALDI-MSI Sample Preparation
		3 Methods
			3.1 Vibratome Calibration
			3.2 Tissue Preparation
			3.3 Vibratome Sectioning
			3.4 Incubation with Isotope-Labeled Nutrients
			3.5 MALDI-MSI Sample Preparation
			3.6 MALDI-MSI Data Acquisition
			3.7 MALDI-MSI Data Preprocessing
		4 Notes
		References
Part IX: Data Processing, Analysis, and Bioinformatics
	Chapter 29: Extracting Knowledge from MS Clinical Metabolomic Data: Processing and Analysis Strategies
		1 Introduction
		2 Materials
			2.1 Availability of the Algorithms
			2.2 Biological Background
		3 Methods
			3.1 Pre-acquisition Normalization
			3.2 MS Acquisition
			3.3 MS Signal Processing
				3.3.1 Peak Detection and Deconvolution
				3.3.2 Alignment
				3.3.3 Data Quality Assessment and Data Cleaning
				3.3.4 Post-acquisition Normalization
				3.3.5 Data Scaling
			3.4 Multivariate Statistics
				3.4.1 Unsupervised Analysis
				3.4.2 Supervised Analysis
			3.5 Metabolite Identification
				3.5.1 Annotation Levels
				3.5.2 Annotation Using MS and MS/MS Information
				3.5.3 Annotation Using Other Molecular Properties from the Separation
			3.6 Retrospective Analysis
		4 Notes
		References
	Chapter 30: Application of a Computational Metabolomics Workflow for the Diagnosis of Inborn Errors of Metabolism in a Laborat...
		1 Introduction
		2 NGMS Analytical Setup
		3 NGMS Computational Workflow
			3.1 Stage 1: Quality Control
			3.2 Stage 2: Data Transfer and Conversion
			3.3 Stage 3: Data Preprocessing and Statistical Analysis
			3.4 Stage 4: Data Interpretation
		4 Notes
		References
Index