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

Importance of the Topic

Matrix assisted laser desorption ionization immunohistochemistry (MALDI-IHC) combines targeted antibody labeling with mass spectrometry imaging (MSI) to map proteins in tissue sections. This approach offers high multiplex capacity, spatial resolution, and the ability to analyze formalin fixed paraffin embedded (FFPE) specimens. Its implementation on affordable benchtop MALDI-TOF systems enables wider adoption in research and clinical settings, supporting studies of cellular interactions and disease biomarkers.

Study Objectives and Overview

This study demonstrates a streamlined MALDI-IHC workflow on Shimadzu benchtop MALDI-8020 and MALDI-8030 instruments using HiPLEX-IHC probes. Two antibody panels (6-plex and 20-plex) were applied to human tonsil FFPE sections at 30 µm lateral resolution. The goals were to evaluate method robustness, signal quality, multiplex performance and its application to detect follicular hyperplasia markers.

Methodology

Sample preparation steps included:

• Mounting FFPE human tonsil sections on poly-lysine coated ITO slides
• Deparaffinization with xylene and graded ethanol washes
• Antigen retrieval in alkaline buffer
• Staining with photocleavable mass tag (PC-MT) labeled antibody probes (6-plex or 20-plex)
• UV-induced tag cleavage
• Deposition of 2,5-dihydroxybenzoic acid (DHB) matrix via iMLayer vapor deposition and recrystallization

Used Instrumentation

• Shimadzu MALDI-8020 and MALDI-8030 EasyCare benchtop linear TOF mass spectrometers
• iMLayer matrix vapor deposition system (Shimadzu)
• IonView software (Shimadzu) for initial data visualization
• IMAGEREVEAL MS software (Shimadzu) for targeted spectral analysis and image generation

Main Results and Discussion

The optimized workflow produced clear isotopic resolution and low background noise. Key findings:

• 6-plex panel: Distinct ion images for CD3ε, CD68, VIM, Col-1A1 and Ki67 with isotopic resolution around m/z 1200–1250.
• 20-plex panel: All 20 peptide mass tags detected within m/z 700–2000 range, validating multiplex capability.
• Matrix sublimation optimization: Best signal-to-noise achieved with 4 min DHB sublimation and 3 min recrystallization at 55 °C (5% IPA).
• Follicular hyperplasia study: Differential Ki67 distribution distinguished normal tissue from hyperplastic germinal centers, correlating with H&E staining.

Benefits and Practical Applications

The benchtop MALDI-IHC platform offers:

• Cost-effective entry into high-plex MSI workflows
• Minimal sample requirements suitable for FFPE archives
• Direct integration of immunohistochemistry into MSI for targeted protein mapping
• Rapid data acquisition (4 hours for 6-plex, under 2 hours for 20-plex at 30 µm resolution)

Future Trends and Opportunities

The technique can evolve through:

• Expansion to higher plex panels for comprehensive tissue proteomics
• Integration with high-resolution MALDI-TOF/TOF or Orbitrap platforms
• Automated workflows and AI-driven image analysis for clinical pathology
• Development of novel photocleavable tags and targeted reagents

Conclusion

This work validates the Shimadzu MALDI-8020/8030 benchtop systems as robust platforms for MALDI-IHC imaging. It demonstrates reliable detection of multiple protein targets in FFPE tonsil sections, including pathological markers of follicular hyperplasia. The accessible workflow and software tools support broader adoption in research and diagnostic laboratories.

References

1. Gars et al Ann Diagn Pathol 2020 44 151421
2. Bryant et al Histopathology 2006 Apr 48 5 505 15