MALDI HiPLEX-IHC Imaging of FFPE Human Kidney Tissue at 5 μm utilizing microGRID on timsTOF fleX MALDI-2

Importance of the topic

The ability to map intact proteins within formalin-fixed, paraffin-embedded (FFPE) tissue at high spatial resolution is essential for understanding complex tissue architecture and cellular interactions. Traditional MALDI imaging of intact proteins faces limitations in ionization efficiency and resolution. Combining the MALDI HiPLEX-IHC workflow with microGRID technology on a timsTOF fleX MALDI-2 platform overcomes these challenges, enabling multiplexed protein imaging at 5 µm without artifacts.

Study objectives and overview

This work aimed to demonstrate the performance of MALDI HiPLEX-IHC with microGRID on FFPE human kidney tissue at 5 µm spatial resolution. Two experimental phases were performed:

• Initial evaluation with three antibodies (Vimentin, Histone H2A, ATPase-1A1) at both 20 µm and 5 µm resolutions to compare image quality and spatial detail.
• Extended multiplex with five antibodies including CD68 and Collagen 1A1 to assess complex marker co-localization and correlation with histology.

Methods and instrumentation

Sample preparation followed the MALDI HiPLEX-IHC protocol:

• FFPE kidney sections were heated, dewaxed through a xylene to TBS gradient, and subjected to basic antigen retrieval and blocking.
• Photocleavable peptide-tagged antibodies were incubated overnight at 4 °C.
• Peptide tags were released by UV exposure; CHCA matrix was applied and recrystallized.
• Imaging was conducted on a timsTOF fleX MALDI-2 instrument equipped with microGRID, at both 20 µm and 5 µm raster sizes.
• Data visualization and co-registration with hematoxylin and eosin (H&E) staining were performed using SCiLS Lab software.

Instrumentation used:

• timsTOF fleX MALDI-2 mass spectrometer with microGRID optics
• SCiLS Lab software for image reconstruction and histological annotation

Results and discussion

Initial experiments at 20 µm and 5 µm showed clear molecular maps of Vimentin, Histone H2A, and ATPase-1A1, with the 5 µm images providing superior resolution and artifact-free features. In the extended study, five markers (Vimentin for glomeruli, Histone H2A for nuclei, ATPase-1A1 for proximal tubules, CD68 for macrophages, Collagen 1A1 for fibrillar collagen) were simultaneously imaged. The 5 µm runs matched pathologist-annotated H&E regions, demonstrating precise localization of each protein and minimal signal bleed or striping artifacts.

Benefits and practical applications

This methodology allows:

• High‐throughput visualization of up to 200 intact proteins in a single run.
• Direct correlation of molecular signatures with histological structures.
• Enhanced understanding of cell–matrix and cell–cell interactions in tissue microenvironments.
• Potential applications in QA/QC, biomarker discovery, and translational research.

Future trends and opportunities

Advancements may include further expansion of antibody panels for deeper multiplexing, integration with machine learning for automated histopathological analysis, and adaptation for clinical workflows. The microGRID approach can be applied to other organ systems and disease models to elucidate spatial proteomics at subcellular resolution.

Conclusion

The combination of MALDI HiPLEX-IHC and microGRID on the timsTOF fleX MALDI-2 platform enables artifact-free, multiplexed protein imaging at 5 µm in FFPE tissue. This approach delivers unprecedented spatial detail, facilitating comprehensive studies of tissue biology for research and diagnostic applications.