Targeted Metabolomics Analysis Process

Targeted metabolomics detects a specific set of compounds, which can be analyzed quantitatively or semi-quantitatively through the use of internal standards. The method analyzes metabolites in a particular metabolic pathway and can help us understand the large number of enzymes involved in metabolism as well as the kinetic processes, ultimately revealing their role in a specific metabolic pathway and thus providing further insight into that pathway.

Targeted metabolomics is more targeted compared to non-targeted metabolomics, focusing on a few or several classes of metabolites associated with biological events, such as lipidomics and glycomics.

In targeted metabolomics studies, sample pre-treatment methods need to be optimized to maximize the enrichment of target compounds and reduce the interference of high intensity ions. The range of compounds to be detected is determined in advance of the study, allowing us to discover new interrelationships between metabolites present in a given physiological state.

Figure from Godoy et al., 2020

Sample collection and processing

Targeted metabolomics collects samples in a targeted manner depending on the target of interest. For example, when performing lipidomics, a solvent with good solubility for lipids is used. The quantification of targeted metabolites is based on the quantitative standard curve of standards, so eligible standards need to be prepared.

Instrument analysis

When using LC-MS to accomplish targeted metabolomics analysis, a number of factors need to be considered that have the potential to affect the accuracy of experimental data. Selecting the appropriate ion source, electrospray (ESI) ion source is the most commonly used method for detecting small molecules in LC-MS based metabolomics studies.

ESI is a soft ionization technique that allows mass spectrometry to detect non-volatile, large molecular weight compounds. Its most important advantages are the ability to enable researchers to detect non-volatile compounds without resorting to derivatization to increase volatility, the reduction of fragment ions, and the simplification of spectral interpretation of complex compounds. However, ESI also has some shortcomings, the most prominent one being the strong ion suppression effect when analyzing complex biological samples.

During ionization, ion suppression occurs when different molecules compete for charge, and the efficiency of ionization is related to the chemical properties of the analyte itself. Thus, for a given ion, the observed ion counts will vary due to differences in its co-ionization with other analytes or background ions. Even if no interfering signal is visible in the mass spectrum, ion suppression still exists. Therefore, the entire analysis must be based on the premise that the composition of the compounds in all samples to be tested is approximately the same. When analyzing and comparing, it is very important to select biological samples with the same matrix. For example, it is best not to compare plasma samples with tissue samples.

In targeted approaches, natural and isotopically labeled standards facilitate the identification and quantification of metabolites and reduce false positives. Quantitative metabolomics can be used to establish baseline levels of metabolites in tissues or organisms, for inter-laboratory comparisons, or to define normal versus "perturbed" states of metabolism. The use of isotopically labeled internal standards (IS) can also help to account for matrix-induced ionization effects that affect analytical precision, thereby improving the sensitivity of bioresponse assays.

Reference

1. Godoy, A. T., Eberlin, M. N., & Simionato, A. V. C. (2020). Targeted metabolomics: Liquid chromatography coupled to mass spectrometry method development and validation for the identification and quantitation of modified nucleosides as putative cancer biomarkers. Talanta, 210, 120640.