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Metabolomics - Targeted

Introduction

Targeted metabolomics methodologies seek to measure a specified and known list of metabolites, which are assessed both qualitatively and quantitatively. Such an approach is useful for a broad variety of applications, such as:

  1. Studying pharmacokinetics profiles of drug metabolism.
  2. Monitoring the efficacy of a particular therapeutic strategy, where the focus is on certain metabolites.
  3. Measuring metabolites’ abundances in biochemical pathways across multiple samples and in different perturbation states.

The Method

The FGCZ supports the analysis of targeted metabolites on a wide range of samples, including:

  • Cells
  • Biological fluids
  • Tissues

The main workhorse for targeted metabolomics is LC-MS employing SRM technologies on a Triple-Quadrupole instrumentation. The process involves the following steps:

  1. Development, optimization, and validation of SRM assays using commercially available reference compounds.
  2. Availability of more than 500 compounds in central metabolic pathways at the FGCZ.
  3. Combination of SRM assays with heavy-labelled internal standards or external calibration curves for absolute quantification of a preselected set of metabolites.
  4. Ensuring high reproducibility and sensitivity in the quantification process.

The FGCZ provides:

  • Established methods
  • Method development
  • User access services

The Workflow

  1. User Meeting, project discussion, and design.
  2. Optimized sample extraction.
  3. Calibration curve preparation.
  4. LCMS acquisition, including calibration and quality control samples.
  5. Data processing (Peak integration, RT correlation, S/N evaluation, IS normalization): Quan Browser, Skyline, QuanLynx.
  6. Absolute quantification in concentration values and LOQ/LOD.

The Output

  1. Primary output: LC-MS raw files. These can be inspected by different software packages, including Xcalibur Quan Browser, Trace Finder, or Skyline.
  2. Final output: An EXCEL file containing, per each targeted compound, peak area values and their conversion to concentration values (absolute quantification). The file also contains peak area values of the calibration series, QCs, blanks, and/or normalization versus internal standards.
  3. Result discussion and explanation: Usually, the results will be discussed and explained. In case of resulting publications, method sections can be provided.

Examples of pathways and metabolite classes

Extensions of the listed pathways can be requested. Some examples include:

  • Steroid metabolism
  • Tryptophan pathway
  • Amino acid analysis
  • Glycolysis and TCA cycle and related compounds
  • Prostaglandins
  • Bile acids
  • Short chain fatty acids (SCFA)

References

This content is largely based on information from ETH Zurich functional genomics center https://fgcz.ch/omics_areas.html.

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Maintained by Dylan Lawless. Scientist at EPFL. Resume and Google scholar. RSS feed