Metabolomics: an evolving field

Metabolomics is one of the ‘-omics’ sciences. Of the ‘-omics’ sciences – genomics, transcriptomics, proteomics and metabolomics – it is the only -omics science which directly reflects the underlying state of cells and tissues and their biochemical activity [1]. It has been used to help the diagnosis of metabolic diseases and congenital metabolic disorders [2].

Advances in the field over the past decade have vastly extended the scope of metabolomic applications. New technologies and workflows are helping to make metabolomics a powerful tool for the identification of disease pathologies and for the discovery of new therapeutic targets as well as novel biomarkers.

How do metabolomics studies work?

Metabolomics is the large-scale study of small molecules, known as metabolites, in biological samples such as cells, tissues and organisms.

Metabolites are small biomolecules with a molecular weight typically less than 1,500 Da. As small biomolecules, they are the building blocks of larger structures, including cell membranes, the proteome and the genome [3].

By quantitatively analyzing the levels of specific metabolites in organisms, cells or tissue, it becomes possible to evaluate the phenotype of the system in response to change, for example changes brought about by environmental factors or the introduction of a therapeutic product.

Many factors are required for successful metabolomic studies, including meticulous sample preparation, a comprehensive toolbox of bioinformatics techniques and pioneering instrumentation [4].

Instrumentation

LC–MS has become well established in the field of metabolomics for a number of reasons. Due to the complexity of the biological matrices, ion suppression can take place when MS is used alone. By coupling a liquid chromatography (LC) system to an MS detection platform, metabolites can be separated depending on their elution times, helping to minimize ion suppression. The inclusion of LC can also facilitate metabolite quantification through chromatographic peak analysis [3].

The instrumentation and workflow used for a specific study depends on the research question. For example, untargeted metabolomic studies often require the use of high-resolution mass spectrometry (HRMS).

Untargeted metabolomics

Typically, the goal of an untargeted metabolomic study is to perform metabolic profiling for the entire metabolome present in the system being investigated. Such a study measures the properties of as many metabolites as possible.

In a given system, there is a huge number of metabolites at very low concentrations and with a wide range of physicochemical properties. It is virtually impossible to simultaneously measure the properties of all metabolites present in a system and measuring as many metabolites as possible poses a significant challenge due to the issues outlined above.

Although untargeted studies present a variety of challenges, it is useful to be able to compare biological systems and such studies are often used to generate hypotheses. To maximize the number of metabolites measured, adequate sample preparation and analytical tools are imperative.

The use of HRMS is generally recommended for untargeted studies because the ability to measure accurate mass can help with metabolite identification [4]. Although specific workflows vary, generally an untargeted workflow consists of: profiling, compound identification and interpretation [5]. Profiling consists of sample preparation and data acquisition.

Data acquisition makes use of techniques such as GC–MS, LC–MS, CE–MS and IC–MS depending on the system in question. Once the data has been acquired, statistical analysis is used to uncover significant events.

When the metabolome has been profiled, the next step is to identify the metabolites present. A wide range of spectral databases and libraries can be used to help compound identification.

With the metabolites present identified, all of the gathered data needs to be interpreted in the context of a biological system. Careful analysis of data from untargeted studies can shine light on complex metabolic pathways and help scientists to propose improved biological models.

Targeted metabolomics

In contrast, targeted studies are generally used when studying specific metabolites of interest. Generally, these metabolites have been identified by untargeted studies.

Targeted studies verify and validate defined groups of metabolites across large sample sets [6]. Due to the number of metabolites in the sample sets, high throughput is required. One of the key elements of targeted studies is high analytical reproducibility. Sufficient reproducibility ensures that any variations are caused by biological differences rather than experimental variation.

Various methods are used to quantitate metabolites in targeted metabolomics studies, including selected reaction monitoring, multiple reaction monitoring and parallel reaction monitoring [7,8].

Future metabolomic applications

A significant proportion of early metabolomics research focused on biomarker identification. Unfortunately, however, a very small proportion of this work has been translated to the clinic.

As the field develops it is becoming clearer that metabolomics can be much more widely applied, with the potential to advance our understanding of biology.

Such studies could also have significant implications in the development of novel drug molecules, data could be used to assess everything from the effects of unanticipated target bindings to quality control in the production of biologics.

For a deeper dive into the field of metabolomics, including exclusive interviews, editorials and articles from Bioanalysis journal, check out our Spotlight.

References

[1] The European Bioinformatics Institute. What is metabolomics?
https://www.ebi.ac.uk/training/online/course/introduction-metabolomics/what-metabolomics
[Accessed 10 June 2020].

[2] Clish CB. Metabolomics: an emerging but powerful tool for precision medicine. Cold Spring Harb. Mol. Case. Stud. doi:10.1101/mcs.a000588 (Epub)(2015).

[3] Kim SJ, Kim SH, Kim JH, Hwang S and Yoo HJ. Understanding metabolomics in biomedical resarch. Endocrinol Metab. 31(1) 7–16 (2016).

[4] Fernie AR, Tethewey RN, Krotzky AJ and Willmitzer L. Metabolite profiling from diagnostics to systems biology. Nat. Rev. Mol. Cell. Biol. 5, 763–769 (2004).

[5] Thermo Fisher Scientific. Untargeted metabolomic workflows.
https://www.thermofisher.com/uk/en/home/industrial/mass-spectrometry/mass-spectrometry-learning-center/mass-spectrometry-applications-area/metabolomics-mass-spectrometry/metabolomics-workflows/untargeted-metabolomics-workflows.html.
[Accessed 10 June 2020].

[6] Thermo Fisher Scientific. Targeted metabolomic workflows.
https://www.thermofisher.com/uk/en/home/industrial/mass-spectrometry/mass-spectrometry-learning-center/mass-spectrometry-applications-area/metabolomics-mass-spectrometry/metabolomics-workflows/targeted-metabolomics-workflows.html.
[Accessed 10 June 2020].

[7] Xiao JF, Zhou B and Ressom HW. Metabolite identification and quantitation in LC–MS/MS-based metabolomics. Trends Anal. Chem. 31, 1–14 (2012).

[8] Zhou J, Liu H, Liu Y, Zhao X and Yin Y. Development and evaluation of a parallel reaction monitoring strategy for large-scale targeted metabolomics quantification. Anal. Chem. 88(8) 4478–4486 (2016).