ToxBase: A Multidimensional ToxCast Reference Database for High-Throughput Human Exposome Analysis

High-resolution mass spectrometry (HRMS) is the gold-standard technique for comprehensively profiling chemical exposures in complex human matrices, making it a powerful analytical tool for advancing human exposome research. Yet the scarcity of HRMS reference data, including collision cross-section (CCS) measurements from ion mobility-mass spectrometry (IM-MS) and MS/MS fragmentation spectra, hinders confident structural annotation of chemical exposure agents across laboratories. We therefore developed ToxBase, a multidimensional (m/z, retention time, CCS, MS/MS) reference database for over 2,000 chemicals sourced from the U.S. Environmental Protection Agency's ToxCast chemical library. Built via high-throughput liquid chromatography-ion mobility-tandem mass spectrometry (LC-IM-MS/MS), the ToxBase database comprises 3,598 precursor ions spanning 2,075 unique compounds with excellent precision (98.5% of compounds display interday CCS RSDs < 1%) and strong cross-platform agreement. A high-quality MS/MS reference library of the fragmented precursors was assembled using targeted data-dependent acquisition and DDARawProcessor, a novel data extraction algorithm. When applied to LC-IM-MS/MS data obtained from human plasma, urine, and fecal samples (n = 20 per matrix), ToxBase rapidly enabled 42 high-confidence (Level 1) identifications. The ToxBase database is freely available and compatible with the open-source MS data processing platform Skyline for vendor-agnostic suspect screening workflows, providing a valuable resource for standardized, large-scale exposome analysis.