Towards Cellular Resolution of Tryptic Peptides in Tissue Sections by MALDI MS Imaging: A Focus on Enzyme Application...

Area of Science:

• Proteomics
• Mass Spectrometry Imaging
• Biochemistry

Background:

• Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI imaging) is crucial for untargeted protein localization and identification in tissues.
• Existing MALDI imaging workflows lack combined high lateral resolution, accurate mass detection, efficient tryptic digestion, and reproducibility.

Purpose of the Study:

• To develop a MALDI imaging workflow enabling reproducible imaging of tryptic peptides in mammalian tissue sections with high resolution in both mass and space.
• To address the limitations of current methods by optimizing parameters for enhanced spatial proteomics.

Main Methods:

• Systematic optimization of trypsin spray parameters (number of sprays, enzyme volume, density, flow rate).
• Development of a custom digestion chamber for high-resolution measurements.
• Implementation of MALDI imaging at a 10 μm pixel size.
• On-tissue MALDI-MS/MS for peptide sequencing.

Main Results:

• Achieved reproducible imaging of tryptic peptides at 10 μm pixel size with high mass resolution (R > 100,000 FWHM) and mass accuracy (RMSE < 3 ppm).
• Demonstrated minimal peptide delocalization through optimized trypsin spraying and controlled on-tissue moisture.
• Enabled identification of up to 283 proteins based on LC-MS/MS data and confirmed 16 tryptic peptide sequences via on-tissue MALDI-MS/MS.

Conclusions:

• The developed MALDI imaging workflow significantly enhances spatial resolution and reproducibility for peptide imaging in tissues.
• Optimized on-tissue digestion dynamics are critical for high-resolution MALDI imaging.
• The findings offer transferable insights for various spatial proteomics applications.