The metabolome consists of all the small molecules, or metabolites, which are components and products of biochemical reactions taking place within the body. Given its dynamic nature, metabolomics is a highly complex, yet informative field.
Download the Multi-Omics Playbook here.
Whilst there have been huge technological advances in metabolomics over the last few years, no analytical method can yet measure the full metabolome. Instead, a combination of approaches is used for a wide coverage of the metabolome. High-resolution techniques often employed in metabolomics include mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. Often, certain applications require slight adaptations to these techniques2. In this feature, written using content from our recent Multi-Omics Playbook, we explore the metabolomics workflow and various methodologies.
THE METABOLOMICS WORKFLOW
Within the metabolomics workflow, one must completely block all enzymes and biochemical reactions by quenching metabolic pathways and metabolite isolation. This creates a stable extract with ratios and concentrations of metabolites that reflect the endogenous state. Careful sample collection and metabolite extraction helps maintain analyte concentrations, increase instrument productivity and reduce the analytical matrix effect. Before conducting metabolomic analysis, it is important to recognise which sample collection and storage method is ideal for your metabolites of interest. These processing steps can cause drastic variation, which can ultimately impact your results and biological assumptions. Metabolomics analysis can be targeted or untargeted2.
- Targeted metabolomic profiling (validation-based) identifies and quantifies a relatively low (usually <100), set number, of analytes. Internal standards (IS) are employed to reduce false positives and improve sensitivity through matrix-induced ionisation effects. However, the low number of metabolites identified may lead to wrong assumptions regarding the metabolic pathway of interest.
- Untargeted metabolomic profiling (discovery-based) identifies a more comprehensive number of analytes and provides relative quantification. Validation with an orthogonal approach must also be used. Care must be taken with sample preparation and analytical methods as these will impact the type of metabolites detected. Two approaches for data acquisition can be used, data-dependent acquisition (DDA) and data-independent acquisition (DIA)3.
MASS SPECTROMETRY (MS)
MS is a staple technique in proteomics. However, it is also widely used for metabolomics. For separation techniques, MS may be used with gas chromatography (GC), liquid chromatography (LC) or capillary electrophoresis (CE)2.
- Gas chromatography (GC) is paired with MS (GC-MS) for analysis of small molecular substances (< 650 Daltons).
- Liquid chromatography (LC) is paired with MS (LC-MS) for analysis of complex molecules, as well as non-volatile or thermally labile compounds with a high molecular weight that cannot be identified by GC-MS. Advances on LC-MS include high-performance LC (HPLC)4, ultra-performance LC (UPLC)5 and ultra-high-pressure LC (UHPLC)4.
- Capillary electrophoresis (CE) is paired with MS (CE-MS) to analyse polar and charged metabolites. CE-MS also enables the profiling of the metabolism of (sub)microliters of samples.
Importantly, MS may also be paired with separation-free techniques for metabolomics analysis. For rapid, accurate and highly repeatable analysis, direct injection or infusion into high –resolution and –accuracy MS (direct infusion MS, DI-MS) can be employed6. This technique does not allow the separation of isomeric compounds, but contamination of the ion source is prevalent6. To analyse metabolites in situ, one can use mass spectrometry imaging (MSI)7. Figure 1 depicts the workflow for MS-based metabolomics approaches, including information on ion sources and analysers2. Of note, certain techniques are most applicable to analyse specific metabolites, especially in the context of polarity2. One should consider this when designing a metabolomics experiment. Figure 1 overviews the preferential techniques for the analysis of specific metabolites.
FIGURE 1. MS TECHNIQUES FOR METABOLOMICS. (a) The stages of the MS workflow and the various techniques, (b) The most suitable metabolomics techniques to analyse specific metabolites with distinct polarities. Figure from Danzi et al, 20231.
NUCLEAR MAGNETIC RESONANCE (NMR) SPECTROSCOPY
NMR spectroscopy is an alternative method for metabolomics analysis. The technique measures the chemical shifts of atomic nuclei with non-zero spin dependent on the atom environment for a particular analyte, for example 1H, 31P or 13C. It allows detection and exploration of the analyte structure. Importantly, NMR spectroscopy allows the user to characterise new compounds and requires little of the sample preparation that is required for MS2,8.
References:
1. Danzi, F. et al. To metabolomics and beyond: a technological portfolio to investigate cancer metabolism. Signal Transduction and Targeted Therapy 8 (2023).
2. Di, S-C. et al. DEPDC1 as a metabolic target regulates glycolysis in renal cell carcinoma through AKT/mTOR/HIF1α pathway. Cell Death & Disease 15 (2024).
3. Schrimpe-Rutledge, A.C., Codreanu, S.G., Sherrod, S.D. and McLean, J.A. Untargeted Metabolomics Strategies – Challenges and Emerging Directions. Journal of the American Society for Mass Spectrometry 27, 1897-1905 (2016).
4. Forcisi, S., Moritz, F., Kanawati, B., Tziotis, D., Lehmann, R. and Schmitt-Kopplin, P. Liquid chromatography – mass spectrometry in metabolomics research: Mass analyzers in ultra high pressure liquid chromatography coupling. Journal of Chromatography A 1292, 51-65 (2013).
5. Zeki, Ö.C., Eylem, C.C., Reçber, T., Kır, S. and Nemutlu, E. Integration of GC-MS and LC-MS for untargeted metabolomics profiling. Journal of Pharmaceutical and Biomedical Analysis 190, 113509 (2020).
6. Ubhi, B.K. Direct Infusion-Tandem Mass Spectrometry (DI-MS/ MS) Analysis of Complex Lipids in Human Plasma and Serum Using the Lipidyzer™ Platform. Methods in Molecular Biology 1730, 227-236 (2018).
7. Buchberger, A.R., DeLaney, K., Johnson, J. and Li, L. Mass Spectrometry Imaging: A Review of Emerging Advancements and Future Insights. Analytical Chemistry 90, 240-265 (2018).
8. Marion, D. An introduction to biological NMR spectroscopy. Molecular & Cellular Proteomics 12, 3006-3025 (2013).
