Targeted Metabolomics Workflows
Targeted metabolomics is quantitative and derives its information from discovery experiments, literature and/or clinical observations before a model or hypothesis is tested.
Testing involves verification and validation of defined groups of known metabolites across large sample sets. These experiments require accuracy, high throughput and reliability. For large scale targeted profiling, using a high resolution accurate mass (HRAM) workflow is optimal, while for routine quantitation, selected reaction monitoring (SRM) on a triple quadrupole MS is the preferred solution.
Large scale targeted profiling
In the targeted metabolomics workflow, analytical or technical reproducibility is key to experimental success. High analytical reproducibility means that findings are due to actual biological variance. It also minimizes experimental replicates, so a smaller number of samples need to be analyzed.
Large scale targeted profiling offers a new perspective on targeted quantitation when compared against the gold standard of quantitation using SRM and triple quadrupole MS. Using HRAM mass spectrometry, targeted analysis can be performed using a method called parallel reaction monitoring (PRM) . This method offers greater selectivity, confirmation of data with MS/MS and retroactive data analysis. There is also less method development.
There are three steps in a targeted profiling workflow:
- Metabolite candidate list creation and method optimization—Ideal fragment ions are chosen from discovery experiments. Collision energy values are adjusted and retention times confirmed.
- PRM data acquisition—Multiple simultaneous fragmentations are generated and analyzed.
- Data analysis—Quantitation is performed using the extracted ion chromatographs (XIC) of each metabolite. Statistical analysis tools are used for biological insight into samples derived from experiment groups. High resolution MS/MS data are collected and are used to identify metabolites when the parent ions cannot be distinguished.
-  Zhou, J., Liu, H., Liu, Y., Liu, J., Zhao, X. and Yin, Y., 2016.
Development and Evaluation of a Parallel Reaction Monitoring Strategy for Large-Scale Targeted Metabolomics Quantification
Analytical Chemistry, 88(8), pp.4478-4486
Routine targeted metabolomics
For routine quantitation of well characterized metabolites, SRM using triple quadrupole MS is the gold standard. The advantages of this workflow include reduced interference, increased detection sensitivity, and streamlined method creation and data acquisition. SRM transitions that are developed from targeted metabolites generate the best signals.
There are three steps in a routine targeted quantitation workflow:
- Metabolite candidate list creation and method optimization—Each metabolite requires determination of its unique diagnostic fragment ions, optimal MS collision energy, authentic standards and a calibrated quantitation method.
- SRM data acquisition—Batches of samples are analyzed using an optimized method complete with appropriate controls, standards and blanks.
- Data analysis—Quantitation and reporting software is utilized during data analysis, wherein calibration curves, ion ratios and peak integrations can be monitored. Usually, data analysis occurs in real-time, so samples that do not match given user criteria are flagged. Report templates typically come as standard.