MR-SP<sup>2</sup>: A Microreactor-Based Workflow for Few-Cell Spatial Proteomics on the Legacy Zeiss PALM MicroBeam

Laser capture microdissection (LCM) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables spatial proteomics at the few-cell level but is constrained by cumulative losses during specimen capture, surface adsorption during processing, and sample transfer prior to LC-MS/MS analysis. The capture-associated losses are particularly relevant for pressure catapulting systems such as the legacy Zeiss PALM MicroBeam, which, despite discontinuation, remains in active use and therefore requires compatible low-loss workflows. We present MR-SP² (microreactor-based sample preparation for spatial proteomics), a one-pot workflow integrating reproducible Zeiss LCM-cut specimen capture, processing with minimized adsorptive losses, and pipetting-free transfer with Evotip disposable precolumns. The workflow was evaluated using a formalin-fixed paraffin-embedded (FFPE) murine kidney tissue analyzed by timsTOF flex LC-MS/MS analysis. Across 50,000 μm³ regions (22 cells), MR-SP² modestly improved proteome depth (3381 ± 80 versus 3174 ± 59 proteins). Decreasing sample input further accentuated the advantage of MR-SP² in maintaining higher identification rates, highlighting the successful reduction of the adsorptive losses. At 12,500 μm³ (5-6 cells), identifications increased to 1145 ± 188 versus 302 ± 126. At 3125 μm³ (1-2 cells), identifications reached 695 ± 112 versus 206 ± 51. MR-SP² improves identification depth for few-cell FFPE samples by nearly 3-fold compared to conventional tube-based workflows.