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

Importance of the Topic

Multiplexed imaging of intact proteins in tissue sections enables detailed spatial proteomics critical for biomarker discovery, disease research, and clinical diagnostics. MALDI-IHC overcomes spectral overlap limitations of fluorescence methods by using photo-cleavable peptide tags and mass detection, allowing simultaneous localization of dozens of protein targets without enzymatic digestion that can compromise spatial fidelity.

Objectives and Study Overview

This work evaluates the compatibility and performance of MALDI-IHC on a SELECT SERIES MRT MALDI-TOF mass spectrometer. Human FFPE tonsil and clear cell renal cell carcinoma (ccRCC) tissues were stained with up to six antibody-tag probes (e.g., CD3ε, CD68, VIM, Collagen-1A1, PanCK, Ki67) to assess imaging accuracy, spatial resolution at 50 µm and 20 µm pixel sizes, and background suppression.

Methodology

Sections were deparaffinized, rehydrated, antigen-retrieved, blocked, and incubated with peptide-tagged antibodies. After UV cleavage of tags, CHCA matrix was applied via HTX M5 sprayer. MALDI imaging was performed in positive mode over m/z 50–2400 with a fixed quadrupole at 1000 Da, 2 kHz laser repetition, and scan speed of 10 s/s. Data were visualized using Waters HDI software.

Used Instrumentation

• Waters SELECT SERIES MRT MALDI-TOF mass spectrometer with multi-reflector ToF analyzer
• HTX M5 matrix sprayer following AmberGen Miralys HiPLEX-IHC workflow
• Chlorohydroxycinnamic acid (CHCA) matrix
• Waters HDI imaging software for data processing

Main Results and Discussion

Imaging of human tonsil at 50 µm demonstrated clear distributions of Ki67, PanCK, Collagen-1A1, VIM, CD68, and CD3ε consistent with literature reports. In ccRCC tissue, Actin-aSM highlighted vascular smooth muscle, VIM marked endothelial filaments, PanCK localized epithelium, and Ki67 identified proliferating nuclei. At 20 µm pixel size, individual Ki67-positive nuclei and micro-structures in Actin-aSM patterns were resolved with minimal background. Mass resolution exceeded 200,000 FWHM and mass accuracy better than 500 ppb, ensuring unambiguous tag identification.

Benefits and Practical Applications of the Method

• High-plex (>40 potential targets) spatial mapping of intact proteins without digestion
• Preserved tissue morphology and antigen localization
• Reduced background and spectral overlap compared to fluorescence
• Applicability to pathology research, biomarker validation, and QA/QC workflows

Future Trends and Potential Applications

Integration of MALDI-IHC with other omics modalities (lipidomics, metabolomics) will enable comprehensive multi-omic spatial studies on a single platform. Development of new photo-cleavable tags and higher-throughput protocols could support larger marker panels and clinical translational research in personalized medicine.

Conclusion

This study confirms that MALDI-IHC on a SELECT SERIES MRT MALDI-TOF instrument provides high-resolution, high-accuracy multiplexed imaging of intact protein targets in FFPE tissues. The method offers robust sensitivity, low background noise, and potential for extensive spatial proteomics applications.

References

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