APPLICATION NOTEBOOK - MS IMAGING AND AMBIENT IONIZATION-MS FOR METABOLOMICS AND LIPIDOMICS

Significance of the Topic

The spatial mapping of metabolites and lipids directly in biological tissues provides critical insight into disease mechanisms, drug distribution, biomarker localization, and food quality. Surface-based desorption ionization approaches such as MALDI, DESI, and DART, combined with ion mobility separation, enable label-free, high-throughput imaging of complex samples under ambient or vacuum conditions. By preserving tissue integrity and avoiding lengthy extraction or chromatography steps, these techniques capture native biochemical distributions that are typically lost in bulk analysis.

Objectives and Study Overview

This compilation of application notes demonstrates how modern ionization and analytical platforms can be used to:

• Discover and visualize biomarkers in tissue sections using high-definition MALDI imaging with multivariate data analysis (PCA, PLS-DA, OPLS-DA).
• Improve spatial resolution, sensitivity, and acquisition speed on the MALDI SYNAPT G2-Si HDMS system.
• Perform untargeted structural identification of lipids on single sections via data-independent MALDI Imaging HDMS^E.
• Measure collision cross sections of lipids and peptides in situ during MALDI imaging.
• Implement ambient-pressure DESI imaging with dual polarity and sequential scans at different resolutions.
• Achieve real-time lipidomic profiling of foods and biofluids by DART-IMS-MS.

Methodology and Instrumentation

Key platforms and methods described include:

• MALDI SYNAPT G2/G2-Si HDMS™ and Xevo® G2-XS Q-Tof with T-Wave™ ion mobility and High Definition Imaging (HDI™) software.
• DESI imaging sources for positive/negative ion mode ambient-pressure analysis; TM Sprayer and manual nebulizers for matrix application.
• DART ion source coupled to IMS-TOF for direct, in-situ ionization of lipids in oils, sebum, and tissues.
• Software suites: MassLynx® for control, apex3D for peak detection, HDI MALDI for image reconstruction, EZInfo™ for multivariate statistics, SIMLipid and LipidMaps for lipid identification, MassFragment™ for MS/MS annotation, MarkerLynx™ XS and HDMS Compare for comparative profiling, and DriftScope™ for mobility visualization.
• Typical acquisition parameters: MALDI mass range 100–3,000 Da, laser repetition rates up to 2.5 kHz, spatial resolutions 15–150 µm; DESI solvent flows 1–1.5 µL/min, spatial resolutions 50–200 µm; DART temperatures 50–450 °C, acquisition times <10 s.

Main Results and Discussion

• Integration of multivariate analysis directly into HDI MALDI software accelerates the discovery of time- and dose-dependent biomarkers in tumor xenografts, enabling rapid ROI definitions and PCA/OPLS-DA correlations to display discriminating ion images.
• The new laser on the SYNAPT G2-Si HDMS yields sharper beam profiles (<30 µm), reducing background noise and enhancing signal-to-noise at sub-50 µm pixels, facilitating detailed lipid mapping in brain tissues.
• Data-independent MALDI Imaging HDMS^E acquires low- and elevated-energy functions in a single run; two-step drift-time and spatial correlation securely matches fragments to precursors for on-tissue lipid identification without targeted MS/MS.
• Collision cross-section (CCS) measurements of phospholipid standards and endogenous lipids directly from sections conform to literature values, demonstrating the ability to probe gas-phase conformations during imaging.
• DESI imaging supports multimodal workflows including dual polarity scans and sequential imaging at different resolutions on the same section, preserving tissue for post-analysis H&E staining.
• DART combined with IMS-TOF separates isobaric lipids on the millisecond scale, generating 3D drift-time versus m/z maps within seconds. Comparative profiling with HDMS Compare distinguishes edible oils and human sebum based on key fatty acids and ceramides.

Benefits and Practical Applications

These integrated approaches offer:

• High-resolution spatial metabolomics and lipidomics in biomedical research, pathology, and pharmacology.
• Accelerated biomarker discovery and validation without serial sectioning or targeted MS/MS design.
• Non-destructive, ambient imaging enabling follow-up histology or additional surface analyses.
• Rapid, real-time compositional screening for food quality control, cosmetics, forensics, and clinical diagnostics.
• Enhanced specificity and sensitivity through gas-phase ion mobility separation of isobaric and isomeric species.

Future Trends and Potential Uses

Emerging directions include:

• Fusion of multimodal imaging (MALDI, DESI, DART) with optical and elemental mapping for holistic tissue phenotyping.
• AI-driven analytics to automate feature selection and biomarker classification in complex MSI datasets.
• Super-resolution and 3D imaging to chart microenvironmental gradients of metabolites and lipids in tumors and organs.
• Intraoperative mass spectrometry guidance via real-time DESI or DART probes for surgical decision-making.
• Point-of-care lipidomic profiling of biofluids by portable IMS-MS devices.

Conclusion

The convergence of advanced ionization techniques (MALDI, DESI, DART), ion mobility separation, and integrated informatics platforms (HDI, DriftScope, EZInfo, HDMS Compare) has revolutionized tissue and surface imaging. Researchers can now acquire high-definition, multiplexed molecular maps, perform rapid structural identification, and execute comprehensive comparative studies with minimal sample preparation. These capabilities are driving forward metabolomics and lipidomics discovery across biomedicine, food science, and beyond.

References

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3. Ridenour WB, Kliman M, McLean JA, Caprioli RM. Structural Characterization of Lipids via MALDI Traveling-Wave Ion Mobility-MS. Anal Chem. 2010;82(5):1881–1889.
4. Yew JY, Cody RB, Kravitz EA. Cuticular Hydrocarbon Analysis by DART-TOF MS in Awake Flies. Proc Natl Acad Sci USA. 2008;105(20):7135–7140.
5. Dear GJ, Munoz-Muriedas J, Beaumont C, et al. Investigating Metabolite Structures with Ion Mobility and Molecular Modelling. Rapid Commun Mass Spectrom. 2010;24(21):3157–3162.