COLLISION CROSS SECTION ENABLED DESI ION MOBILITY MASS SPECTROMETRY IMAGING

Importance of the Topic

Mass spectrometry imaging (MSI) enhanced with ion mobility spectrometry (IMS) enables spatial mapping of molecules while providing structural information via collision cross section (CCS). This approach addresses challenges in complex samples such as tissues or drug delivery systems by separating ions based on size and shape prior to mass analysis, increasing confidence in compound identification without the need for chromatography.

Objectives and Study Overview

• Introduce a workflow to derive CCS values directly from desorption electrospray ionization (DESI) high-definition MS (HDMS) imaging data.
• Benchmark experimental CCS measurements against known standards and literature values to validate accuracy.
• Demonstrate the ability to resolve isobaric species and characterize endogenous lipids in mouse brain tissue sections.

Methodology and Instrumentation

• Ion Source: DESI on a modified 2D stage delivering a 95:5 methanol:water spray with 0.1% formic acid at 3 µL/min, 4.5 bar nebulizing N₂, 2.5 kV sprayer voltage in positive mode.
• Mass Spectrometer: SYNAPT G2-Si ion mobility QToF configured for traveling wave IMS (TWIMS) separation, mass range 50–1200 m/z, pixel size 100 µm, 0.5 s per scan.
• Calibration Standards: Leucine-enkephalin lock mass, Major Mix ion mobility CCS mix, and multiple CCS reference compounds spotted on a well plate.
• Data Processing: MassLynx 4.2 for acquisition, HDI 1.4 for image reconstruction and peak fitting, CCS calculation in Excel using drift time bins and linear calibration parameters.

Main Results and Discussion

• CCS values derived from DESI HDMS imaging matched literature and reference standards with deviations below 2%.
• Ion mobility effectively separated isobaric peaks (same accurate mass but different CCS), which would otherwise require ultrahigh mass resolution to resolve.
• In mouse brain tissue imaging, numerous endogenous lipids and metabolites were detected and their CCS values determined, many of which align with or extend existing lipid databases.

Benefits and Practical Applications

Combining DESI MSI with ion mobility and CCS measurement:

• Enhances molecular specificity by adding a structural dimension to mass-based identification.
• Resolves isobaric interferences without chromatographic separation.
• Supports high-throughput spatial profiling in pharmaceutical, clinical, and QA/QC laboratories.

Future Trends and Potential Applications

• Integration of large CCS databases and machine learning models for automated identification.
• Coupling MS/MS imaging with IMS to provide fragmentation and structural insights in one experiment.
• Expansion to single-cell imaging and in vivo tissue analysis.
• Standardization of IMS workflows for broader adoption in metabolomics and lipidomics.

Conclusion

A streamlined workflow for calculating CCS from DESI HDMS imaging has been established, demonstrating robust agreement with known standards and enabling resolution of complex isobaric species. This method enhances the analytical power of MSI, offering a valuable tool for spatially resolved structural characterization.

Reference

1. Waters Pub. #720004176EN, April 2012.
2. Bush, M. F.; Hall, Z.; Giles, K.; Hoyes, J.; Robinson, C. V.; Ruotolo, B. T. Anal. Chem. 2010, 82, 9557.
3. Pagel, K.; Natan, E.; Hall, Z.; Fersht, A. R.; Robinson, C. V. Angew. Chem. Int. Ed. 2013, 125, 379.
4. Paglia, G.; et al. Anal. Chem. 2015, 87, 1137.
5. Paglia, G.; et al. Anal. Chem. 2014, 86, 3985.