Chapter 1
State-of-the-art in LCâMS Approaches for Probing the Polar Metabolome
Artemis Lioupi1a,b,c, Maria Marinaki1a,b,c, Christina Virgilioua,b,c, Helen Gika,b,c,d, Ian Wilson,e and Georgios Theodoridisa,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 (GCâMS), liquid chromatography coupled to MS (LCâMS), and capillary electrophoresis coupled to MS (CEâMS), are widely and effectively utilized for the analysis of microbial, plant, and animal metabolomes.3â5 Comprehensive two-dimensional (2D) techniques (LC Ă LC, GC Ă 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 LCâMS.15,16 Yet, such analysis is often much less sensitive compared to LC and GCâMS-based techniques, although this depends on the nature of the analyte. GCâMS 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. CEâMS is highly effective for the analysis of polar and charged metabolites and can offer important and complementary information, supplementing that obtained by LCâMS 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 LCâMS 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 LCâMS 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 acidâbase 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 LCâMS 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 Îźm particle diameter for packed columns or in the range of 2.6â2.8 Îźm 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...