High-speed MALDI-TOF/TOF imaging of mouse brain tissue performed on intact proteins and after on-tissue digestion

Significance of the Topic

High-resolution MALDI mass spectrometry imaging (MSI) enables spatial mapping of intact proteins and peptides directly in tissue, revealing molecular distributions critical for understanding biological processes and disease states.
The dual workflow—top-down proteoform imaging and bottom-up peptide imaging following on-tissue digestion—provides complementary data on protein modifications and identities, improving the depth and specificity of tissue analysis.

Objectives and Study Overview

This study demonstrates the application of a Bruker rapifleX MALDI-TOF/TOF platform for high-speed imaging of fresh-frozen mouse brain tissue in both intact protein (top-down) and tryptic peptide (bottom-up) modes.
Key goals include comparing spatial segmentation of proteoforms and peptides, evaluating imaging resolution and acquisition speed, and validating peptide identifications via on-tissue MS/MS.

Methodology

• Tissue Preparation
  • Fresh-frozen mouse brain sagittal sections (10 µm) mounted on conductive slides and vacuum-dried.
  • Intact protein sections washed with a Carnoy protocol; peptides generated by on-tissue tryptic digestion under controlled humidity and temperature.
• Matrix Application
  • Sinapinic acid for intact proteins; α-cyano-4-hydroxycinnamic acid (HCCA) for digested peptides.
  • Applied using HTX TM-sprayer with optimized flow rates, number of passes, and track spacing.
• Data Acquisition
  • Bruker rapifleX TOF/TOF in positive linear mode for m/z 2,000–24,000 (intact proteins) and positive reflector mode for m/z 600–2,400 (peptides).
  • Pixel size of 30 µm, Single laser focus, spectra accumulated from 400–600 laser shots per pixel.
  • On-tissue MS/MS without collision gas for peptide sequence confirmation.
• Data Analysis
  • SCiLS Lab software with total ion current normalization and unsupervised spatial segmentation.
  • Receiver operating characteristic (ROC) analysis to identify region-specific features.
  • Database searches (MASCOT) for peptide identification from on-tissue MS/MS spectra.

Used Instrumentation

• Bruker rapifleX MALDI-TOF/TOF system
• HTX TM-sprayer connected to isocratic LC and syringe pumps
• Leica CM 1900 UV cryotome
• Reflecta MF-5000 slide scanner
• SCiLS Lab software for image processing

Main Results and Discussion

• Top-Down Imaging
  • Spatial segmentation distinguished cerebellum and cortex based on proteoform patterns.
  • ROC analysis identified histone variants (H1, H2A/B, H3, H4) with distinct localization and modification profiles in cerebellum.
  • RapifleX achieved 30 µm pixel size and ~14× faster acquisition compared to previous instruments.
• Bottom-Up Imaging
  • On-tissue tryptic digestion produced peptide images reflecting anatomical regions; segmentation isolated cerebellum cluster.
  • MS/MS identified peptide ARTKQTAR from histone 3.3; its ion image matched top-down distribution.
  • MS/MS imaging of fragment ion [b+18]7 further confirmed peptide localization.
• Complementarity of Workflows
  • Top-down reveals intact proteoform distributions and modification patterns.
  • Bottom-up and on-tissue MS/MS enhance mass accuracy, resolution, and peptide specificity, resolving isobaric species.

Benefits and Practical Applications

• High-definition mapping of protein modifications and distributions in complex tissues.
• Ability to differentiate tissue subtypes and disease stages by molecular signatures.
• Enhanced acquisition speed and spatial resolution facilitate large-scale studies.
• On-tissue MS/MS enables direct peptide identification without off-line digestion.

Future Trends and Possibilities

High-speed MALDI imaging platforms will likely integrate multi-omics workflows, combining lipidomics, metabolomics, and proteomics in a single experiment.
Advances in AI-driven data analysis and machine learning will improve segmentation, classification, and biomarker discovery.
Applications may extend to clinical diagnostics, drug distribution studies, and personalized medicine by mapping molecular heterogeneity in patient tissues.

Conclusion

The Bruker rapifleX MALDI-TOF/TOF system offers a versatile imaging platform for top-down and bottom-up workflows, enabling high-resolution, high-speed mapping of proteoforms and peptides in tissue.
The complementary approaches provide detailed insights into protein modifications, localization, and identity, advancing research in biomedical and clinical fields.

References

1. Rauser S., Marquardt C., Balluff B., et al. J. Proteome Res. 2010, 9(4), 1854–1863.
2. Lahiri S., Sun N., Solis-Mezarino V., et al. Proteomics 2016, 16, 437–447.
3. Alexandrov T., Becker M., Deininger S.-O., et al. J. Proteome Res. 2010, 9(12), 6535–6546.