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

Comprehensive xenometabolome characterization is essential for understanding the effects of xenobiotics in biological systems. This study presents a multidimensional analytical workflow integrating orthogonal chromatographic separations, trapped ion mobility spectrometry (TIMS), high-resolution mass spectrometry and biotransformation-informed data processing to address xenometabolome assessment challenges. Zebrafish larvae exposed to 4-Methylbenzotriazole (4-MeBT) were used as a challenging case study. TIMS dimension provided orthogonal experimental evidence for isomer annotation, with inverse reduced mobility (1/K₀) supporting conjugation site assignment for the dominant O-S-4MeBT and O-G-4MeBT isomers. The combination of TIMS with the Parallel Accumulation Serial Fragmentation (PASEF) acquisition further reduced spectral complexity, enhanced signal-to-noise ratio, and improved MS/MS coverage (70%), generating high-quality analytical evidence crucial for structural elucidation. To leverage these analytical dimensions, we developed a data processing strategy that leverages in-silico-based suspect screening and biotransformation-informed nontarget screening. In this regard, we introduce two novel frameworks; the "Building Blocks" (BB) concept which interprets unknown bio-TPs as modular assemblies of parent- and pathway-derived substructures, and the "Spectral Characteristics Knowledgebase" (SCKB), which use known biotransformation MS/MS motifs to provide structural insights and facilitate unknown identification. Our results demonstrated the identification of all previously known 4-MeBT bio-TPs with enhanced confidence (O-Sulfate- and O-Glucuronide-4MeBT) and the discovery of 29 new bio-TP features across 12 bio-TP classes, highlighting its efficacy in unraveling complex xenobiotic metabolism. Among these, a putative dimerization product (4-MeBT-263) was reported for the first time in zebrafish. Overall, this workflow has the potential to advance the understanding of bio-TP formation and detoxification processes in xenometabolome studies.