Trapped ion mobility spectrometry (TIMS) adds an additional separation dimension to mass spectrometry (MS) imaging, however, the lack of fragmentation spectra (MS2) impedes confident compound annotation in spatial metabolomics. Here, we describe spatial ion mobility-scheduled exhaustive fragmentation (SIMSEF), a dataset-dependent acquisition strategy that augments TIMS-MS imaging datasets with MS2 spectra, where fragmentation experiments are systematically distributed across the sample and scheduled for multiple collision energies per precursor ion. Extendable data processing and evaluation workflows are implemented into the open source software MZmine. The workflow and annotation capabilities are demonstrated on a rat brain tissue thin section, measured by matrix-assisted laser desorption/ionisation (MALDI)-TIMS-MS, where SIMSEF enabled on-tissue compound annotation through spectral library matching and rule-based lipid annotation within MZmine and mapped the (un)known chemical space by molecular networking. The SIMSEF algorithm and data analysis pipelines are open source and modular to provide a community resource.
Supplementary information: On-tissue dataset-dependent MALDI-TIMS-MS2 bioimaging
Supplementary information containing additional figures and algorithm description. Updated with author information.