MALDI Mass Spectrometry Imaging Reveals Distinct Spatio-Molecular Phospholipid Distributions in Mouse Lungs

Importance of the topic

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) enables spatially resolved mapping of lipid species directly on tissue sections. This approach is essential for understanding the heterogeneous distribution of phospholipids in the lung, which underlie key physiological functions and pathological processes such as asthma, fibrosis, cancer, and infection.

Objectives and study overview

This study aimed to develop a workflow to preserve three-dimensional lung architecture for high-resolution MALDI MSI and on-tissue tandem MS analysis. The goals were to characterize baseline phospholipid distributions in healthy mouse lungs using both positive and negative ion modes and to establish a reference lipid atlas for future pulmonary disease investigations.

Methodology and instrumentation

Lung tissues from normal athymic nude mice were inflated with gelatin, embedded, frozen, and cryosectioned at 10 μm onto indium tin oxide (ITO) slides. Matrices were applied by an HTX TM sprayer: 2,5-dihydroxybenzoic acid for positive ion mode and 1,5-diaminonaphthalene for negative ion mode. Imaging was performed on a Bruker rapifleX MALDI TOF/TOF instrument with the following parameters:

• Positive ion mode: 20 μm raster size, 500 laser shots per pixel
• Negative ion mode: 50 μm raster size, 200 laser shots per pixel
• On-tissue MS/MS imaging in negative reflectron mode on adjacent sections

Data were processed using flexImaging for visualization and SCiLS Lab software for unsupervised spatial segmentation. Post-imaging H&E staining confirmed morphological correlations.

Main results and discussion

Positive ion images revealed that sodiated phosphatidylcholine PC(32:0) (m/z 756.6) localizes around alveolar regions, potassiated PC(34:2) (m/z 796.5) surrounds bronchioles and trachea, and heme (m/z 616.2) appears centrally. Spatial segmentation accurately distinguished lobes, airways, and vasculature based on lipid signatures.

Negative ion imaging and MS/MS profiling identified distinct distributions of phosphatidylinositols (PIs), phosphatidylglycerol (PG), and phosphatidic acid (PA). Key assignments included PI(22:6/18:0) and PI(20:4/20:2) at m/z 909.5 in muscle and alveoli; PI(20:4/18:0) at m/z 885.5 broadly distributed; PI(18:2/18:0) at m/z 861.5 in muscle; PG(18:0/20:4) at m/z 797.5 and PA(O-18:1/18:1) at m/z 687.5 in connective tissue.

On-tissue MS/MS imaging of the isobaric parent ion m/z 857.6 resolved four PI isomers—PI(20:4/16:0), PI(18:2/18:2), PI(18:0/18:4), and PI(18:1/18:3)—each exhibiting unique localization patterns across lung periphery, muscle, connective tissues, and parenchyma.

Benefits and practical applications

• Provides high-resolution, multiplexed lipid maps of healthy lung tissue
• Links lipid distribution patterns with anatomical structures via automated segmentation
• Enables differentiation of isobaric lipids directly on tissue without extraction
• Establishes a reference lipid atlas for comparative studies in pulmonary disease models

Future trends and potential applications

Future developments may integrate MALDI MSI with quantitative analyses, three-dimensional reconstruction, and multimodal imaging (e.g., immunostaining or fluorescence). Applying this platform to models of tuberculosis, asthma, fibrosis, and lung cancer can uncover disease-specific lipid biomarkers and therapeutic targets. Advances in instrumentation and data analytics will enhance spatial resolution, sensitivity, and throughput.

Conclusion

MALDI MSI combined with on-tissue MS/MS offers a powerful approach for detailed mapping of phospholipid distributions in mouse lungs. The established protocol preserves tissue architecture and delivers high spatial and molecular resolution, providing a foundational atlas for future pulmonary lipidomics and disease research.

References

1. Scott AJ, Chandler CE, Ellis SR, Heeren RMA, Ernst RK. Maintenance of deep lung architecture and automated airway segmentation for 3D mass spectrometry imaging. Scientific Reports. 2019;9:20160.
2. Fahy E, Sud M, Cotter D, Subramaniam S. LIPID MAPS online tools for lipid research. Nucleic Acids Research. 2007;35:W606-W612.