MR-SP²: A Microreactor for Upward Pressure-Catapulting Laser Microdissection for Mass Spectrometry-Based Spatial Proteomics at Single-Cell Resolution

ABSTRACT Laser capture microdissection (LCM) combined with liquid chromatography-tandem mass-spectrometry (LC–MS/MS) enables spatially resolved proteomics at few-cell scale, yet losses from minute LCM-cut specimens, particularly with upward pressure-catapulting systems and subsequent processing, limit depth and reproducibility. We present MR-SP² (Microreactor-based Sample Preparation for Spatial Proteomics), a one-pot-in-solution workflow that integrates reproducible LCM-cut specimen capture, processing with minimized adsorptive losses, and pipetting-free transfer with Evotip disposable precolumns. The workflow is demonstrated on a formalin-fixed paraffin-embedded (FFPE) murine kidney using upward pressure-catapulting LCM for targeted isolation of defined tissue specimens. Across 50,000 µm³ regions (22 cells), MR-SP² modestly improved proteome coverage (3,381 ± 80 versus 3,174 ± 59 proteins). Decreasing sample input further accentuated the advantage of MR-SP² in maintaining higher identification rates, highlighting the successful reduction of adsorptive losses of the MR-SP² workflow. At 12,500 µm³ (5-6 cells), identifications increased to 1,145 ± 188 versus 302 ± 126. At 3,125 µm³ (1-2 cells), identifications reached 695 ± 112 versus 206 ± 51. MR-SP² improves identification depth for few-cell FFPE samples nearly threefold and provides an LCM-compatible preparation that expands the robustness, applicability of upward pressure-catapulting LCM within the spatial proteomics toolkit. Competing Interest Statement The authors have declared no competing interest.