A Multidimensional Workflow for Comprehensive Xenometabolome Profiling by Integrating TIMS-PASEF with LC-HRMS and...

Area of Science:

• Environmental Chemistry and Toxicology
• Analytical Chemistry
• Metabolomics

Background:

• Understanding xenobiotic effects requires comprehensive xenometabolome characterization.
• Assessing xenometabolomes is challenging due to complex biological systems and diverse chemical structures.
• Existing methods struggle with isomer identification and structural elucidation of xenobiotic metabolites.

Purpose of the Study:

• To develop and validate a multidimensional analytical workflow for enhanced xenometabolome assessment.
• To improve the identification and structural elucidation of xenobiotic biotransformation products (bio-TPs).
• To investigate the metabolism of 4-Methylbenzotriazole (4-MeBT) in zebrafish larvae.

Main Methods:

• Integrated orthogonal chromatography, trapped ion mobility spectrometry (TIMS), and high-resolution mass spectrometry.
• Employed Parallel Accumulation Serial Fragmentation (PASEF) for improved MS/MS data acquisition.
• Developed novel data processing strategies: Building Blocks (BB) concept and Spectral Characteristics Knowledgebase (SCKB).

Main Results:

• TIMS provided orthogonal evidence for isomer annotation and conjugation site assignment (O-S-4MeBT, O-G-4MeBT).
• PASEF enhanced MS/MS coverage to 70%, facilitating structural elucidation.
• Identified all known 4-MeBT bio-TPs with high confidence and discovered 29 new bio-TP features across 12 classes, including a novel dimerization product (4-MeBT-263).

Conclusions:

• The developed multidimensional workflow significantly advances xenometabolome characterization capabilities.
• The BB concept and SCKB frameworks offer powerful tools for interpreting unknown xenobiotic metabolites.
• This approach enhances the understanding of xenobiotic biotransformation and detoxification pathways.