Title:Optimization of Pseudotargeted Metabolomics: Fully Integrating the
Advantages of Both Targeted and Untargeted Approaches
Volume: 22
Issue: 7
Author(s): Yong Li, Tao Pang*, Junli Shi, Zhaoli Xu, He Xie, Ge Bai, Xuejun Chen and Lu Zhao
Affiliation:
- Tobacco Chemistry Center, Yunnan Academy of Tobacco Agricultural Sciences, Kunming, Yunnan, 650021, China
Keywords:
Pseudotargeted, metabolomics, chromatography, mass spectrometry, multiple reaction monitoring, identification.
Abstract:
Introduction: This study aimed to address persistent challenges in pseudotargeted metabolomics,
particularly the limited compatibility with diverse sample types, by developing an enhanced
method integrating the strengths of targeted and untargeted approaches.
Methods: An upgraded pseudotargeted metabolomics method was developed, incorporating a sample-
specific MS-RI library (SSMSRIL) to identify novel metabolites in new samples. Newly discovered
metabolites were dynamically added to a pseudotargeted MRM list. Additionally, MRM transitions
for 227 target metabolites were integrated, resulting in a final method monitoring >500 metabolites.
This design facilitates the extraction and addition of new metabolites to the monitoring list. The
method was established and evaluated using gas chromatography-tandem mass spectrometry (GCMS/
MS).
Results: Evaluation with new samples revealed that 33-40% of all detected metabolites were identified
exclusively through the integrated targeted MRM transitions. This demonstrated their significant
role in expanding metabolite coverage. Furthermore, 23-54% of metabolites detected in new sample
types were absent from the initial SSMSRIL list.
Discussion: The substantial proportion (23-54%) of metabolites detected in new sample types missing
from the original library underscores the critical necessity of dynamically updating the pseudotargeted
MRM list when applying the method to new samples. This update mechanism is vital for maintaining
broad metabolite coverage and method applicability across diverse sample matrices.
Conclusion: The enhanced pseudotargeted method significantly improves metabolite coverage and
adaptability to new sample types through dynamic MRM list updating and the integration of targeted
MRM transitions. While developed using GC-MS/MS, the core concept is readily transferable to
liquid chromatography (LC)-based full-scan and MRM methodologies, broadening its potential impact.
