Multiplexed Targeted Imaging of Intact Proteins in Tissue by MALDI-IHC, on the SELECT™ SERIES MRT

Importance of the Topic

Mass spectrometry imaging (MSI) of intact proteins in tissue sections has long been challenged by low ionization efficiency, high molecular weight of proteins, and interference from endogenous signals. Multiplexed MALDI-immunohistochemistry (MALDI-IHC) overcomes these limitations by using photo-cleavable peptide tags linked to antibodies, enabling targeted, high-plex imaging of proteins directly on tissue without digestion. This approach preserves spatial localization, reduces background noise, and supports superior multiplexing compared to fluorescence immunohistochemistry.

Objectives and Study Overview

This study demonstrates the compatibility and advantages of MALDI-IHC when coupled with the SELECT SERIES™ MRT MALDI-ToF mass spectrometer. Key goals included:

• Validating tag identification accuracy using high mass resolution and accuracy.
• Comparing image quality at 50 μm and 20 μm pixel sizes.
• Visualizing four clinically relevant proteins in human kidney clear cell renal carcinoma (ccRCC) tissue.

Methodology

Sample preparation followed established workflows for formalin-fixed paraffin-embedded (FFPE) tissues. Human tonsil sections pre-tagged for six markers validated instrument performance at 50 μm. Human ccRCC grade 3 sections were tagged in-house for VIM, Actin-aSM, PanCK, and Ki67. After antibody binding, UV exposure cleaved photo-cleavable tags. Matrix coating used α-cyano-4-hydroxycinnamic acid applied by HTX™ M5 sprayer, followed by recrystallization.

Instrumentation

• SELECT SERIES MRT MALDI-ToF mass spectrometer: orthogonal acceleration, quadrupole, multi-reflecting TOF analysis yielding >200,000 FWHM resolution and <500 ppb mass accuracy.
• HTX™ M5 matrix sprayer for uniform matrix deposition.
• AmberGen Miralys™ workflow for sample tagging and UV photocleavage.
• Waters™ High Definition Imaging (HDI™) software for data processing and visualization.

Main Results and Discussion

Validation on human tonsil yielded clear localization of six protein markers matching reference data. In ccRCC samples, MALDI-IHC at 50 μm resolved distinct distributions: Actin-aSM in vessel walls and tumor margins; VIM enriched at tumor border indicating inflammatory modulation; PanCK confined to healthy epithelium confirming non-epithelial tumor origin; Ki67 highlighting proliferative tumor nuclei. Increasing spatial resolution to 20 μm sharpened features, allowing potential single-cell level localization of Ki67 signals. Extracted ion chromatograms and resolved isotopic profiles demonstrated negligible background interference and unambiguous tag identification, underscoring the high confidence afforded by the instrument’s resolving power.

Benefits and Practical Applications

• Direct visualization of intact proteins without digestion preserves spatial fidelity.
• High sensitivity and specificity through low background and mass accuracy.
• Superior multiplexing (theoretical >100-plex) enables comprehensive molecular phenotyping on a single section.
• Rapid workflow compatible with clinical and research laboratories for biomarker mapping and disease characterization.

Future Trends and Applications

The integration of MALDI-IHC with high-resolution MSI promises multi-omics spatial profiling—combining lipids, metabolites, and proteins in one experiment. Advances in tag design and instrument throughput will expand plex capacity and enable subcellular resolution. Clinical adoption may drive new diagnostics in oncology, immunology, and precision medicine.

Conclusion

This study confirms that coupling MALDI-IHC with the SELECT SERIES MRT system delivers high-confidence, multiplexed imaging of intact proteins in tissue sections. The approach offers high spatial resolution, minimal background, and robust tag identification, paving the way for advanced spatial proteomics and translational applications in biomedical research.

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