Understanding cellular heterogeneity in neurodegenerative diseases requires metabolic profiling with high molecular coverage and spatial resolution. While MALDI-MSI has advanced spatial metabolomics, its sensitivity towards many analytes, including metabolites, remains limited due to low ionisation efficiencies, restricting metabolite coverage and spatial resolution.
Post-ionisation (PI) technologies overcome these limitations by enhancing ionisation efficiencies and molecular coverage. PI introduces a secondary ionisation event, spatially and temporally separated from the initial desorption/ionisation process. Here, we outline the capabilities and applications of two PI methods implemented on Orbitrap and timsTOF instruments.
MALDI-2, the most established PI technique, employs a secondary UV laser to intercept the MALDI plume, significantly enhancing detection of lipids, glycans, peptides, and pharmaceuticals. We implemented MALDI-2 on an Orbitrap mass spectrometer, demonstrating significant increases in sensitivity and coverage for metabolite imaging in non-neurological (kidney) and neurological tissues (spinal cord), including detection of metabolites not observed with conventional MALDI. This added sensitivity enables higher spatial resolution through oversampling, achieving imaging at pixel sizes several times smaller than the laser spot diameter. We also explored single-cell metabolomics of iPSC-derived astrocytes, detecting and imaging metabolites, such as nucleotides, following fluorescence microscopy—representing the first MALDI-based study of low-molecular-weight metabolites from single cells.
Using a prototype timsTOF coupled with plasma PI, we applied quantitative MSI (Q-MSI) to investigate lipid changes in the SOD1-G93A ALS mouse model. With an optimized internal standard mixture applied before matrix deposition, Q-MSI was performed at 20 µm pixel size to assess disease-related lipid composition changes in spinal cord tissues.
This work expands MALDI-MSI capabilities for investigating metabolic and lipidomic alterations in neurological diseases at single-cell spatial resolution.