An Automated and Fast Sample Preparation Workflow for Laser Microdissection Guided Ultrasensitive Proteomics

Area of Science:

• Proteomics
• Mass Spectrometry
• Cellular Biology

Background:

• Spatial tissue proteomics links cellular phenotypes to proteome states.
• Current methods require optimization for large patient cohorts and low sample inputs.
• Streamlined, loss-minimized workflows are crucial for broader application.

Purpose of the Study:

• To introduce an automated, streamlined sample preparation protocol for laser microdissected tissues.
• To enable efficient proteome analysis from low-input archival samples.
• To facilitate spatial proteomics in large-scale studies and single-cell applications.

Main Methods:

• Automated sample preparation using the cellenONE robotic system for laser microdissected samples.
• Optimized protocol for lysis, formalin de-crosslinking, and tryptic digest.
• Seamless integration with Evosep ONE LC system for 'on-the-fly' sample clean-up.

Main Results:

• Successful validation in human tonsil archival tissue.
• Profiling of proteomes in spatially-defined B-cell, T-cell, and epithelial microregions (4000 μm²).
• Achieved depth of ~2000 proteins with high cell type specificity.

Conclusions:

• The developed automated protocol is efficient and suitable for low-input archival tissue samples.
• This method enhances the applicability of spatial proteomics to large patient cohorts.
• Detailed guidelines and equipment templates are provided for accessibility.