Benchtop Matrix Assisted Laser Desorption Ionization Mass Spectrometry Imaging of Human Tonsil Proteins using MALDI HiPLEX-IHC Probes

Significance of the Topic

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) enables spatial mapping of lipids, metabolites, peptides and proteins within tissue sections. Coupling MALDI with immunohistochemistry probes bearing photocleavable mass tags (MALDI-IHC) extends this capability to targeted protein visualization. This approach supports advanced tissue phenotyping, disease research and potential clinical diagnostics.

Study Objectives and Overview

The study demonstrates the feasibility of high-plex protein imaging on formalin-fixed paraffin-embedded (FFPE) human tonsil sections using a benchtop MALDI-TOF instrument (Shimadzu MALDI-8020/8030). Two multiplexed antibody panels—a 6-plex for general immune markers and a 20-plex oncology probe panel—were applied at 30 μm spatial resolution to evaluate performance, throughput and biological insights.

Methodology

Sample preparation involved poly-L-lysine coated ITO slides, deparaffinization, graded ethanol washes and antigen retrieval. Sections were incubated with AmberGen photocleavable mass-tagged antibodies and coated with 2,5-dihydroxybenzoic acid (DHB) matrix via sublimation. Optimal DHB recrystallization (55 °C, 5 % IPA) was established for spectral quality. MALDI imaging was performed in linear mode on the benchtop MALDI-8020 with a 30 μm step size. Data acquisition and processing utilized IonView and IMAGEREVEAL MS software.

Instrument Configuration

• MALDI-8020 and MALDI-8030 benchtop MALDI-TOF mass spectrometers
• iMLayer sublimation device for DHB matrix application
• AmberGen photocleavable mass-tagged antibodies (6-plex and 20-plex panels)
• IonView and IMAGEREVEAL MS software for data extraction and visualization

Key Results and Discussion

• 6-plex panel: 60 201 spectra acquired in ~4 h 8 min yielded isotopically resolved ion images correlating with germinal centers and T-cell distributions; overlays matched H&E and conventional IHC features.
• 20-plex oncology panel: 27 300 spectra in ~1 h 52 min enabled detection of all 20 mass tags across the extended mass range (~2 kDa) with clear structural mapping.
• Follicular hyperplasia sample: 29 650 spectra in ~2 h 2 min revealed distinct Ki67 and CD3ε distributions compared to normal tonsil, highlighting potential clinical biomarker differentiation.

Benefits and Practical Applications

The use of an entry‐level benchtop MALDI‐TOF platform offers a cost‐effective, robust solution for multiplex protein imaging. High sensitivity and spatial resolution support research in immunology, oncology and pathology. Rapid acquisition times facilitate higher throughput workflows suitable for QA/QC and potential translational studies.

Future Trends and Opportunities

• Expansion of probe multiplexing capacity beyond 20 targets for comprehensive proteomic mapping.
• Improved spatial resolution via advanced optics and stage control for subcellular imaging.
• Integration with digital pathology and machine learning for automated image analysis and biomarker discovery.
• Quantitative MALDI-IHC for absolute protein quantification in clinical samples.

Conclusion

This study confirms that benchtop MALDI-TOF MSI coupled with photocleavable mass‐tagged antibodies can reliably image up to 20 protein targets at 30 μm spatial resolution in FFPE tissue. The method aligns closely with traditional histological assessments and opens avenues for high-plex tissue phenotyping in research and diagnostic settings.

References

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