Advanced Mass Spectrometry-based Analytical Separation Techniques for Probing the Polar Metabolome

Rawi Ramautar

• Publisher: Royal Society of Chemistry
• Year: 2021
• eBook ISBN: 9781839163531
• Edition: 1
• Topic: Physical Sciences
• Subtopic: Biochemistry

The efficient analysis of polar and charged metabolites in biological samples remains a huge challenge in the field of metabolomics. Over the past years, novel mass spectrometry-based analytical tools have been developed to enable the sensitive and efficient profiling of polar ionogenic metabolites in various biological samples. This book gives the reader a comprehensive overview of these recent technological developments.

Topics covered include the use of chemical labelling strategies for allowing the analysis of polar metabolites using reversed-phase liquid chromatography–mass spectrometry (RPLC-MS) and the latest methodological developments in RPLC-MS, hydrophilic interaction liquid chromatography (HILIC)-MS and ion-pair LC-MS approaches. Attention is also paid to developments in nano-LC-MS and capillary electrophoresis–mass spectrometry methods specifically for profiling polar metabolites in small volume biological samples. The utility of ion-mobility MS and NMR spectroscopy will also be outlined. Sample preparation is the key part in the analytical workflow employed for metabolomics. Therefore, ample emphasis will be given on recent solid-phase extraction and solid-phase micro-extraction methods. Finally, analytical techniques for chiral metabolic profiling will also be considered.

Discussing the state-of-the-art of the proposed topics in one single book for probing the polar metabolome, using relevant examples, is unique and needed in the metabolomics field. This book has relevance and appeal to an international audience of analytical and biomedical researchers in industry and academia.

Chapter 1

State-of-the-art in LC–MS Approaches for Probing the Polar Metabolome

Artemis Lioupi^1a,b,c, Maria Marinaki^1a,b,c, Christina Virgiliou^a,b,c, Helen Gika,^b,c,d, Ian Wilson,^e and Georgios Theodoridis^a,b,c

^a Laboratory of Analytical Chemistry, School of Chemistry, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece;

^b Biomic AUTh, Center for Interdisciplinary Research and Innovation (CIRI-AUTH), Balkan Center B1.4, 10th km Thessaloniki-Thermi Rd, P.O. Box 8318, GR 57001, Thessaloniki, Greece;

^c FoodOmicsGR Research Infrastructure, AUTh Node, Center for Interdisciplinary Research and Innovation (CIRI-AUTH), Balkan Center B1.4, 10th km Thessaloniki-Thermi Rd, P.O. Box 8318, GR 57001, Thessaloniki, Greece;

^d Department of Medicine, Aristotle University, 54124 Thessaloniki, Greece;

^e Division of Computational and Systems Medicine, Department of Metabolism, Digestion and Reproduction, Imperial College, South Kensington, London, United Kingdom Email: [email protected]

1.1 Introduction: Metabolomics and the Polar Metabolome

Metabolomics is a rapidly growing research area that focuses on the systematic study of small molecule metabolites found in various types of biological samples, such as tissues, cells, and body fluids.^1,2 These small molecules (molecular masses typically below 1000 amu) have diverse physicochemical properties and are present over a wide range of concentrations, from fmol to mM, rendering global analysis a challenging task. Recent advances in analytical techniques partially address the challenges related to standardizing metabolomics and maximizing the amount of extracted information. Nuclear magnetic resonance (NMR) spectroscopy and MS-based techniques, such as gas chromatography coupled to mass spectrometry (GC–MS), liquid chromatography coupled to MS (LC–MS), and capillary electrophoresis coupled to MS (CE–MS), are widely and effectively utilized for the analysis of microbial, plant, and animal metabolomes.^3–5 Comprehensive two-dimensional (2D) techniques (LC × LC, GC × GC) or tandem-MS (MS/MS) have also been tested in the quest to obtain the most detailed profiles of metabolites in a single analysis. More recently ion mobility-based techniques have also been integrated into metabolic and lipidomic phenotyping protocols to enhance metabolome coverage.⁶

Considering the scope of the analysis, metabolomics studies are widely categorized into two major approaches: targeted and untargeted metabolomics.⁷ Targeted methods aim at the identification and quantification of a small subset of pre-selected molecules (from the low tens to some hundreds of analytes).^8,9 Targeted methods are extensively used in clinical and medical applications and find use in the confirmation of biomarkers and hypothesis testing. Analysis can be carried out in a quantitative or semi-quantitative manner with the use of internal standards, surrogate matrices, or surrogate analytes. These approaches exploit the extensive understanding of a wide range of metabolic enzymes, their kinetics, end products, and the known biochemical pathways to which they contribute.¹⁰ Tandem triple quadrupole instruments (TQ) operating in multiple reaction monitoring (MRM) mode have enabled simultaneous targeted analysis of hundreds of metabolites involved in important metabolic pathways.¹¹ Untargeted metabolomics, on the other hand, focuses on the detection and identification of as many metabolites as possible in a sample, to obtain accurate and comprehensive metabolic fingerprints.¹² A recent review covered the topic of targeted metabolomics.⁵ Untargeted metabolomics relies mainly on either ¹H NMR spectroscopy, direct infusion (DI) MS, or the hyphenation of high-efficiency chromatographic separations with the high-sensitivity and high-resolution mass spectrometry analysis on time of flight (TOF) or Orbitrap-MS detectors in full scan mode.¹³ Ultimately, untargeted experiments offer wide metabolome coverage, whereas targeted experiments provide better quantification, usually through the usage of isotope-labeled internal standards and defined conditions for the mass spectrometer and the chromatographic separation system.¹⁴

Even after the continuous improvement and refinement of analytical platforms over the past few years, the analytical research community is still struggling with the analysis of highly polar and charged metabolites. A potential solution is resorting to ¹H NMR spectroscopy, where the polarity of the metabolite is irrelevant, and only the concentration and the possession of protons to generate a measurable signal are required. ¹H NMR spectroscopy requires minimum sample preparation and is especially suited for the analysis detection and characterization of polar metabolites such as sugars, organic acids, alcohols, polyols, and other highly polar compounds that are less tractable to LC–MS.^15,16 Yet, such analysis is often much less sensitive compared to LC and GC–MS-based techniques, although this depends on the nature of the analyte. GC–MS also presents important limitations in the analysis of highly polar compounds, due to their thermolability and low volatility.¹⁷ Therefore, a derivatization step is required before analysis. CE–MS is highly effective for the analysis of polar and charged metabolites and can offer important and complementary information, supplementing that obtained by LC–MS regarding the biological composition of samples.^18,19 However, CE currently lacks the robustness required for analyzing large sets of biological samples, and its use in the field of metabolomics is still relatively limited.²⁰

Here, we describe state-of-the-art in LC–MS approaches for the analysis of the polar metabolome. There are very strong needs to map the polar metabolome because polar hydrophilic metabolites are important in many basic biochemical pathways. With the ever-growing need to explain altered cellular metabolism in cancer and other diseases, the evaluation of polar central carbon metabolites associated with energy production has been at the forefront of metabolomics.²¹ Similarly, amino acid metabolism as a whole or specific pathways, e.g., the tryptophan pathway, is increasingly screened in disease biomarker discovery. Continuous enhancements in LC separations, MS technologies, bioinformatic tool development, and database expansion have significantly improved the LC–MS workflow and offer new perspectives on the analysis of polar compounds.²¹

1.2 LC Technologies: General Introduction

HPLC can resolve relatively complex analyte mixtures that contain analytes with various molecular weights, polarities, and acid–base properties. To accomplish this, different modes of separation, such as reversed-phase chromatography (RPLC), normal-phase liquid chromatography (NPLC), ion-exchange chromatography (IEC), and hydrophilic interaction (HILIC) chromatography have been developed. The selection of the appropriate LC mode relies on the analytes and the sample of interest, as well as the available analytical platforms.^22,23

The combination of the detection capabilities of a mass spectrometer, especially when linked to an LC separation provides advanced analytical capabilities, such as high sensitivity, increased specificity, and rapid analysis. Hence LC–MS is now the most powerful instrumental platform for the analysis of non-volatile molecules and in particular small molecule metabolites.²⁴ Important trends are seen in the use of ultra-high performance (UHPLC), multi-dimensional (MD) LC, and technical aspects, such as miniaturization/microfluidics, and new MS interfaces.²³ The establishment of commercially available UHPLC has been used to dramatically increase the coverage of the metabolome and/or throughput in comparison to regular HPLC methods.²⁵ UHPLC typically uses stationary phases of sub-2 μm particle diameter for packed columns or in the range of 2.6–2.8 μm for fused-core particles and high solvent flow rates and pressures, of up to 19 000 psi. Higher flow rates lead to shorter analytical run times and increased peak capacity. The greater chromatographic resolution of UHPLC compared to HPLC leads to reduced matrix effects and a reduction of mass spectral overlap. Capillary (Cap) LC and nano-LC could also be listed among the current topics of interest in liquid chromatography²⁶ as they offer increased sensitivity and much lower solvent consumption. New column technologies are conti...

Table of contents

• Cover
• Title
• Copyright
• Contents
• Chapter 1 State-of-the-art in LC–MS Approaches for Probing the Polar Metabolome
• Chapter 2 Chemical Derivatization for Polar Metabolome Analysis
• Chapter 3 Ion Pair Liquid Chromatography–Mass Spectrometry for Probing the Polar Metabolome
• Chapter 4 Hydrophilic Interaction Chromatography–Mass Spectrometry (HILIC–MS) Approaches for Probing the Polar Metabolome
• Chapter 5 Advances in Solid Phase Microextraction (SPME) for Metabolomics
• Chapter 6 State-of-the-art Capillary Electrophoresis Mass Spectrometry Methods for Analyzing the Polar Metabolome
• Chapter 7 Ion Mobility–Time-of-flight Mass Spectrometry and Applications for Metabolomics
• Chapter 8 NMR Approaches for Probing the Polar Metabolome
• Chapter 9 State-of-the-art Mass Spectrometry-based Approaches to Explore the Polar Metabolome: Alzheimer's Disease as a Case Study
• Chapter 10 Analytical Techniques for Material-limited Metabolomics: Recent Developments and Applications
• Chapter 11 More with Less: Single-cell Metabolomics by Mass Spectrometry
• Chapter 12 Chiral Metabolomics
• Subject Index