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-SP{superscript 2} (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 {micro}m3 regions (22 cells), MR-SP{superscript 2} modestly improved proteome coverage (3,381 {+/-} 80 versus 3,174 {+/-} 59 proteins). Decreasing sample input further accentuated the advantage of MR-SP{superscript 2} in maintaining higher identification rates, highlighting the successful reduction of adsorptive losses of the MR-SP{superscript 2} workflow. At 12,500 {micro}m3 (5-6 cells), identifications increased to 1,145 {+/-} 188 versus 302 {+/-} 126. At 3,125 {micro}m3 (1-2 cells), identifications reached 695 {+/-} 112 versus 206 {+/-} 51. MR-SP{superscript 2} 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.