Metabolomics is a collective term for the biochemical reactions within an organism, serving as the material foundation for maintaining life and a crucial basis for studying life activities. Metabolomics, based on high-throughput analysis and bioinformatics technologies, investigates endogenous metabolic activities of life under internal and external influences. This includes the detection and analysis of the types, quantities, and patterns of changes in metabolites, thereby exploring the essence of collective life activities' occurrence and development.
Research indicates that various diseases, including cancer, as well as liver, kidney, cardiovascular, and neurological diseases, are associated with physiological disruptions or cellular functional loss caused by changes in intracellular metabolic states. Metabolomics has become a powerful tool in the post-genomic era of functional genomics, serving as a significant means for the large-scale screening of new biomarkers for early prediction, diagnosis, and classification of diseases. It is an essential technique in precision medicine, with its research focusing on small molecules with a relative molecular mass less than 1000. Metabolites include primary metabolites such as sugars, amino acids, and fatty acids, as well as secondary metabolites like phenols, terpenes, flavonoids, alkaloids, and sulfur-containing compounds.
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Advantages of Metabolomics
1. The types and quantity changes of metabolites are easy to detect.
2. Compared to genomics and proteomics, the technical methods are simpler.
3. In comparison to genomics and proteomics, metabolites are fewer in number, making them easy to detect, validate, and analyze.
4. Changes in metabolic levels can instantly reveal the physiological and pathological status of the organism.
Classification of Metabolomics
From the perspectives of research purposes and methods, metabolomics is typically classified into two types: untargeted metabolomics and targeted metabolomics. Additionally, in recent years, a "quasi-targeted" metabolomics approach, which combines the advantages of both untargeted and targeted methods, has also seen some development. Untargeted Metabolomics refers to the unbiased detection of dynamic changes in all small-molecule metabolites (mainly endogenous small molecules with a relative molecular mass of less than 1000 Da) in cells, tissues, organs, or organisms before and after stimulation or disturbance. This is achieved using LC-MS, GC-MS, NMR technologies. The approach involves screening differential metabolites through bioinformatics analysis, conducting pathway analysis on these different metabolites, and revealing the physiological mechanisms underlying their changes. Targeted Metabolomics, on the other hand, focuses on the research and analysis of a specific class of metabolites.
What are the characteristics of these two strategies, and which one should be chosen for research? This article will discuss the differences between these two strategies from the aspects of experimental design, instrument platforms, and implementation processes.
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Experimental Design
Targeted metabolomics is characterized by its clear experimental expectations, as it specifically aims to detect and analyze key metabolites within a particular metabolic pathway or a specific class of compounds. For instance, in the context of tumor research, where the glycolytic pathway is commonly activated, a study focusing on a specific tumor mechanism might emphasize the glycolysis pathway. Targeted metabolomics, in this case, provides a focused and precise approach to address such hypotheses.
To illustrate further, consider the example of acyl-CoA metabolites, which have the potential to modify proteins. Conducting targeted metabolomics to specifically analyze changes in acyl-CoA metabolites allows researchers to uncover variations related to protein acylation modifications. This targeted approach proves beneficial when the goal is to delve into the intricacies of specific metabolic pathways and their implications in cellular processes.
In contrast, untargeted metabolomics is the preferred strategy when there is no clear experimental expectation or when the interest spans across all metabolic processes. This approach involves unbiased detection and analysis of dynamic changes in all small-molecule metabolites, providing a comprehensive view of the metabolome without focusing on specific pathways. Untargeted metabolomics is particularly useful in exploratory studies where researchers aim to uncover novel insights or patterns within the entirety of the metabolic landscape.
Untargeted versus targeted metabolomics studies (Schrimpe-Rutledge et al., 2016).
Instrument Platforms
In targeted metabolomics, precise quantification is essential, necessitating instrument platforms adept at quantitative research. The primary tools employed are triple quadrupole mass spectrometers coupled with liquid chromatography or gas chromatography. These instruments allow for accurate quantification of specific metabolites, making them well-suited for targeted studies aiming to identify key metabolites within particular metabolic pathways.
On the other hand, untargeted metabolomics places a significant emphasis on qualitative analysis. High-resolution mass spectrometers, such as those of the qTOF or Orbitrap classes, are commonly utilized in conjunction with liquid chromatography. Alternatively, platforms based on Nuclear Magnetic Resonance (NMR) may be employed. These instruments provide the high resolution and sensitivity required for the comprehensive detection and characterization of a broad spectrum of metabolites without specific targeting.
In summary, targeted metabolomics leans towards precision and quantification, favoring triple quadrupole mass spectrometers with liquid or gas chromatography. In contrast, untargeted metabolomics places a premium on qualitative insights, utilizing high-resolution mass spectrometers with liquid chromatography or NMR-based platforms to explore the entire metabolome without preconceived targeting.
Implementation Process
In targeted metabolomics, the process involves the utilization of standardized chemical compounds for the target metabolites. Each target compound undergoes method optimization to achieve high sensitivity and specificity. Absolute quantification of individual target compounds in samples is achieved by comparing chromatographic peaks with standard curves generated from standardized chemical compounds. This approach ensures precise and quantitative measurement of specific metabolites through techniques such as triple quadrupole mass spectrometry coupled with liquid or gas chromatography.
In contrast, untargeted metabolomics doesn't require the purchase of standardized chemical compounds. Samples are analyzed under specific conditions, and chromatographic peaks from all samples are arranged. Statistical methods are then employed to identify differences in signals between groups. Subsequently, metabolite identification is carried out using features such as MS, MS/MS, or chemical shifts. Finally, enrichment analysis using databases like GO/KEGG helps discover significantly regulated metabolic pathways.
Both approaches have their advantages and limitations, often complementing each other. They are frequently combined to discover and quantify differential metabolites. This combined approach is valuable in the in-depth study and analysis of subsequent metabolic molecular markers. It plays a crucial role in various fields, including food authentication, disease research, animal model validation, biomarker discovery, disease diagnosis, drug development, drug screening, drug evaluation, clinical research, plant metabolism studies, and microbial metabolism research.
Reference
- Schrimpe-Rutledge, Alexandra C., et al. "Untargeted metabolomics strategies—challenges and emerging directions." Journal of the American Society for Mass Spectrometry 27.12 (2016): 1897-1905.
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