MULTIPLEXED TARGETED IMAGING OF INTACT PROTEINS IN TISSUE BY MULTI REFLECTING TIME OF FLIGHT (MRT) MALDI-IHC

Significance of the Topic

Multiplexed MALDI immunohistochemistry offers a powerful route to spatially resolve multiple intact protein targets in tissue sections without on tissue digestion. By linking cleavable peptide tags to antibodies, this approach overcomes limitations of traditional fluorescence immunohistochemistry, such as spectral overlap and restricted marker capacity, and enables high multiplexing for research and diagnostic applications.

Objectives and Study Overview

This study demonstrates the compatibility and performance advantages of multiplexed MALDI IHC on a multi reflecting time of flight mass spectrometer. Major goals include unambiguous detection of released peptide tags, high mass resolution and accuracy, and preservation of spatial localization across tissue types including human tonsil and renal carcinoma sections.

Methodology and Instrumentation

Sections of formalin fixed paraffin embedded human tonsil and human clear cell renal cell carcinoma were prepared following a standardized protocol comprising deparaffinization, rehydration, antigen retrieval, blocking, and antibody staining with cleavable tags. Tags targeted proteins such as Ki67, PanCK, collagen 1 A1, VIM, CD68, CD3epsilon, Actin aSM. UV light was applied to cleave tags prior to matrix deposition with CHCA using an HTX M5 sprayer.

• Mass spectrometer: SELECT SERIES MRT MALDI-TOF in positive ion mode
• Mass range: 50–2400 Da with fixed quadrupole at 1000 Da
• Laser repetition rate: 2 kHz, scan speed: 10 spectra/sec
• Image acquisition: 50 μm and 20 μm pixel sizes, laser focus 4.0 mm and 5.8 mm respectively

Main Results and Discussion

High mass accuracy (>500 ppb) and resolution (>200000 FWHM) enabled clear separation of tag signals from endogenous ions and minimal background noise. In tonsil sections, distributions of six targets matched established patterns from alternative platforms. In renal carcinoma tissue, tumor morphology and marker localization were visualized at both 50 μm and 20 μm resolution. Actin aSM distinctly highlighted vascular smooth muscle, VIM traced endothelial filaments, PanCK outlined epithelium, and Ki67 marked proliferating nuclei down to individual pixels. Detailed microstructures became evident at 20 μm, demonstrating retention of spatial specificity.

Benefits and Practical Applications

• High multiplexing capacity enables imaging of over 40 markers per section, limited only by tag availability
• Retention of native protein localization avoids artifacts introduced by on tissue digestion
• Low background and high mass resolution support confident target identification
• Potential integration with multi OMIC workflows on the same instrument streamlines spatially resolved analyses

Future Trends and Potential Applications

Advances may include expansion of tag libraries to cover broader proteomes, integration with lipid and metabolite imaging for true multi OMIC spatial mapping, and application to clinical pathology for high dimensional tissue profiling. Further optimization of instrument throughput and automation will facilitate adoption in research and diagnostics.

Conclusion

This work validates multiplexed MALDI IHC on a SELECT SERIES MRT mass spectrometer as a robust method for high resolution, high multiplex protein imaging in intact tissue. The combination of cleavable peptide tags and high mass performance enables detailed spatial proteomics without digestion, paving the way for comprehensive spatial OMIC studies.

References

1 G Yagnik et al J Am Soc Mass Spectrom 2021 32 4 977–988