TIMSImaging: an open and interoperable workflow for trapped ion mobility mass spectrometry imaging data processing and visualization.

summaryMass spectrometry imaging (MSI) enables high-throughput spatial mapping of molecules, but the lack of chromatographic separation limits its utility for complex biological samples. Ion mobility (IM) provides key orthogonal separation. However, open-source tools dedicated for IM-MSI data analysis remain scarce and the integration of ion mobility information into downstream analysis is underexplored. Here, we present TIMSImaging, an open-source workflow for processing and visualization of MALDI-TIMS-MS data from Bruker timsTOF instruments. TIMSImaging incorporates a graph-based two-dimensional feature extraction algorithm for separation of isobaric peaks by ion mobility, supports collision cross section (CCS) calculation, and exports results as imzML files with ion mobility for downstream analysis. We demonstrate its capabilities on three case studies spanning different sample types, analyte types, and downstream tasks. AVAILABILITY AND IMPLEMENTATION: TIMSImaging is released as open-source software under the MIT License. The source code, installation instructions, documentation, and case study Vignettes are available at https://github.com/YinyueZhu/TIMSImaging.