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MR-SP2: A Microreactor-Based Workflow for Few-Cell Spatial Proteomics on the Legacy Zeiss PALM MicroBeam.

Manuel Metzger1,2, Maximilian Maldacker3,4, Tobias Hutzenlaub1,2

  • 1Hahn-Schickard, Georges-Koehler-Allee 103, Freiburg 79110, Germany.

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Summary

A new workflow, MR-SP2, enhances spatial proteomics by minimizing sample loss during laser capture microdissection (LCM) and processing. This method significantly improves protein identification rates, especially for small FFPE tissue samples.

Keywords:
FFPELC-MS/MSLCMfew-cell proteomicssample preparationspatial proteomics

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Area of Science:

  • Proteomics
  • Mass Spectrometry
  • Molecular Biology

Background:

  • Spatial proteomics using laser capture microdissection (LCM) and LC-MS/MS offers few-cell resolution but suffers from sample loss.
  • Legacy Zeiss PALM MicroBeam systems, still in use, require specialized low-loss workflows due to capture-associated losses.

Purpose of the Study:

  • To develop and evaluate MR-SP2, a novel one-pot workflow for spatial proteomics.
  • To minimize sample losses during specimen capture, processing, and transfer for few-cell FFPE samples.

Main Methods:

  • Integration of Zeiss LCM-cut specimen capture with microreactor-based sample preparation (MR-SP2).
  • Minimized adsorptive losses during processing and pipetting-free transfer using Evotip precolumns.
  • Analysis of formalin-fixed paraffin-embedded (FFPE) murine kidney tissue using timsTOF flex LC-MS/MS.

Main Results:

  • MR-SP2 demonstrated improved proteome depth in larger FFPE samples (50,000 μm3).
  • Significantly higher protein identification rates were observed with decreasing sample input, highlighting reduced adsorptive losses.
  • At 3125 μm3 (1-2 cells), MR-SP2 achieved nearly 3-fold higher identifications compared to conventional methods.

Conclusions:

  • MR-SP2 effectively reduces cumulative sample losses in spatial proteomics workflows.
  • The developed workflow enhances proteome identification depth for few-cell FFPE samples.
  • MR-SP2 provides a compatible solution for legacy Zeiss PALM MicroBeam users requiring low-loss protocols.