Lipidomic Imaging of isobaric lipids using high‐resolution ion mobility mass spectrometry with DESI XS

Importance of Lipidomic Imaging

Desorption electrospray ionization (DESI) imaging mass spectrometry enables direct molecular mapping of lipids in tissue sections under ambient conditions. However, complex biological matrices contain numerous lipid species with overlapping mass‐to‐charge ratios, leading to ambiguous assignments. Integrating high‐resolution ion mobility separation addresses this challenge by distinguishing isobaric and closely related lipid ions based on their structural differences.

Study Objectives and Overview

This study aims to combine multi‐pass cyclic ion mobility spectrometry (IMS) with DESI imaging to enhance the specificity and resolution of lipid maps in tissue sections. By applying successive mobility separations in a cyclic high‐resolution IMS device, the authors evaluate the ability to resolve and identify lipid species that share identical or very similar m/z values.

Methodology

• Sample Preparation: Mouse brain sections were thaw‐mounted on glass slides, vacuum dried, and analyzed without additional extraction or labeling.
• DESI Imaging: A Waters DESI‐XS source delivered a methanol–water (98:2) spray with 0.1% formic acid at 2 µL/min and 0.65 kV to ionize surface‐bound lipids.
• Ion Mobility Separation: A quadrupole time‐of‐flight mass spectrometer equipped with a multi‐pass cyclic IMS device collected single‐pass and seven‐pass mobility data over m/z 50–1200.
• Data Processing: MassLynx, DriftScope and HDI software were used to extract, visualize and co‐register ion mobility and spatial imaging data.

Instrumentation

The system comprised a Select Series cyclic IMS Q-ToF instrument featuring:

• A circular 100 cm RF ion guide with over 600 electrodes and T-Waves enabling high‐resolution mobility separation.
• Multi‐pass capability to extend path length and increase resolving power up to ~150 resolution in seven passes.
• Pre‐ and post‐storage arrays allowing selective ion injection, IMSn functionality and ejection of mobility‐filtered ions.

Key Results and Discussion

Multi‐pass IMS markedly improved separation of isobaric lipid ions. In a model m/z 456 window, single‐pass mobility resolved only the largest species, while seven passes separated three distinct drift time populations. For m/z 810.6, initial imaging showed a single peak; after seven passes, two peaks at 92.93 ms and 94.24 ms were resolved. Subsequent MS/MS confirmed one as protonated PC(18:0_20:4) and the other as sodiated PC(18:0_18:1), differing by only 2.4 mDa. Spatial overlays revealed distinct localization patterns for each lipid.

Benefits and Practical Applications

• Enhanced Specificity: Resolving isobaric lipids increases confidence in molecular assignments.
• Improved Spatial Resolution: Mobility‐filtered images reveal distinct tissue distributions of structurally similar lipids.
• Streamlined Workflow: Ambient DESI imaging requires minimal sample prep and is compatible with high‐resolution IMS.

Future Trends and Potential Applications

Further development of cyclic IMS devices may enable higher pass numbers and IMSn experiments, supporting detailed structural elucidation of complex lipid species. Integration with other imaging modalities and informatics tools could advance spatial multi‐omics, biomarker discovery, and clinical diagnostics. Real‐time or in vivo ambient ion mobility imaging also represents a promising frontier.

Conclusion

Combining DESI imaging with multi‐pass cyclic ion mobility separation significantly enhances the resolution and specificity of lipidomic maps in tissue. This approach permits confident discrimination of isobaric lipids, yielding clearer insights into lipid distributions and supporting a wide range of research and QA/QC applications in analytical chemistry.