Routine Workflow of Spatial Proteomics on Micro-formalin-Fixed Paraffin-Embedded Tissues

Area of Science:

• Proteomics
• Spatial Biology
• Biotechnology

Background:

• Formalin-fixed paraffin-embedded (FFPE) tissues are crucial for spatial proteomics.
• Current methods face challenges in achieving high resolution and protein identification efficiency.
• A routine, accessible workflow is needed for spatial proteomics using micro-FFPE tissues.

Purpose of the Study:

• To evaluate and optimize a spatial proteomics workflow for micro-FFPE tissues.
• To establish a routine, device-independent protocol for spatial proteomics.
• To enable high-resolution protein identification from minimal tissue areas.

Main Methods:

• Developed innovative techniques including optimized decross-linking and simplified peptide generation.
• Integrated laser capture microdissection (LCM) with mass spectrometry for protein identification.
• Focused on optimizing the capture rate and balancing peptide generation with excision area.

Main Results:

• Established a routine workflow for spatial proteomics on micro-FFPE tissues.
• Achieved protein identification from a minimized area of 0.002 mm² and consistent analysis from 0.05 mm².
• Successfully constructed a preliminary spatial proteomic atlas of the mouse brain.

Conclusions:

• The developed workflow overcomes key technological challenges in spatial proteomics.
• This method allows for detailed spatial proteomic analysis of FFPE tissues with high resolution.
• The study provides a foundation for creating comprehensive spatial proteomic atlases.