High-performance MALDI imaging analysis of on-tissue digested proteins in mammalian FFPE tissues using the Bruker rapifleX MALDI-TOF/TOF

Significance of the Topic

MALDI mass spectrometry imaging (MSI) of proteins in formalin-fixed, paraffin-embedded (FFPE) tissues unlocks valuable proteomic information from large archival sample collections. Overcoming FFPE-induced cross-linking and paraffin interference is essential to map spatial protein distributions in clinical and research settings.

Objectives and Overview of the Study

This work presents a complete workflow to perform high-resolution MALDI-MSI on on-tissue tryptic digests of various mammalian FFPE sections (human thyroid, human breast cancer, mouse brain) using the Bruker rapifleX MALDI-TOF/TOF. Key goals include:

• Developing an optimized sample preparation protocol (deparaffinization, antigen retrieval, on-tissue digestion, matrix deposition)
• Acquiring peptide imaging data at 20–25 µm lateral resolution
• Applying multivariate analysis (spatial segmentation, PLSA) to reveal region-specific peptide patterns
• Identifying marker peptides by direct MALDI-MS/MS and validating spatial assignments by MS/MS imaging

Methodology

Sample sections (3–6 µm thick) were mounted on conductive slides. Following baking at 85 °C, paraffin removal used graded solvents (xylene, isopropanol, ethanol series, water). Antigen retrieval employed heated water in a decloaking chamber (110 °C, 20 min). On-tissue digestion was performed with porcine trypsin (25 ng/µl) sprayed via HTX TM-sprayer, incubated 2 h at 50 °C under humid conditions. α-Cyano-4-hydroxycinnamic acid matrix was applied with HTX TM-sprayer. MALDI data were acquired on the rapifleX MALDI-TOF/TOF in positive reflector mode at pixel sizes of 20–25 µm and m/z ranges 600–3200. Selected peptides underwent MS/MS directly from tissue without collision gas. Datasets were normalized to total ion count and analyzed in SCiLS Lab for spatial segmentation and probabilistic latent semantic analysis (PLSA).

Used Instrumentation

• Bruker rapifleX MALDI-TOF/TOF mass spectrometer
• HTX TM-sprayer for enzyme and matrix deposition
• NXGEN decloaking chamber (Biocare Medical) for antigen retrieval
• Reflecta MF-5000 scanner and MIRAX DESK (3DHISTECH) for optical imaging
• SCiLS Lab software for multivariate spatial data analysis

Main Results and Discussion

• Spatial segmentation of human thyroid FFPE revealed distinct tissue subtypes based on peptide profiles. Hierarchical trees and mean spectra illustrated five main regions.
• High-resolution imaging of m/z 644.4 in thyroid confirmed that structural features down to 40–50 µm (2 pixels) are resolved.
• In breast cancer sections, segmentation and PLSA independently differentiated fatty/connective versus tumor tissue. Co-registered H&E images corroborated these molecular distinctions.
• Direct MALDI-TOF/TOF MS/MS identified key peptides: collagen α2 (m/z 1562.8), actin cytoplasmic 1 (m/z 1198.7), histone H2A.V (m/z 944.5).
• MS/MS imaging of mouse brain peptide AVFPSIVGRPR (actin) and its fragment ion (m/z 1042.4) demonstrated consistency between MS and MS/MS maps, excluding isobaric interference.

Benefits and Practical Applications

• Enables proteomic mapping in standard clinical FFPE archives
• Integrates high throughput data acquisition (rapifleX) with robust spatial statistics (SCiLS Lab)
• Direct on-tissue MS/MS identification accelerates biomarker discovery
• MS/MS imaging adds specificity, confirming peptide assignments and minimizing artifacts

Future Trends and Possibilities

Advancements may include further miniaturization of pixel size toward cellular resolution, automated sample preparation to reduce delocalization, integration with deep-learning for pattern recognition, expansion to multiplexed ion mobility separation, and routine clinical deployment for pathology-guided proteomics.

Conclusion

A tailored FFPE sample prep workflow combined with the Bruker rapifleX MALDI-TOF/TOF delivers spatially resolved peptide images at 40–50 µm resolution. Multivariate segmentation and direct MS/MS identification enable confident mapping of tissue-specific proteomes. The approach paves the way for high-throughput molecular histology and biomarker research in archived clinical tissues.

References

• Asperger A., Niemeyer D., Henkel C., Ly A., Hecht S., Cornett S. High-performance MALDI Imaging of On-Tissue Digested Proteins in Mammalian FFPE Tissues. Bruker Daltonik GmbH; 2017.
• SCiLS Lab 2017. SCiLS GmbH; Bruker Daltonics.
• Mascot Server. Matrix Science; London, UK.