Novel protocol for spatial proteome analysis of microdissected formalin-fixed paraffin-embedded tissues

Novel In-insert FFPE proteomics combines single glass insert FFPE tissue processing with spatial quantitative data-independent mass spectrometry (DIA). The method represents a potent protocol for spatial FFPE tissue subsection processing in combination with laser capture microdissection, achieving sufficient MS signal from 50 um size mammary acini FFPE voxels. By eliminating additional robotic platforms, desalting and detergent removal steps prior to injection to the liquid chromatograph, this method minimizes protein losses and keep the analysis cost effective. In-insert proteomics maintains sufficient sensitivity to preserve spatial context within a single FFPE tissue slide. This project aim was to acquire data-dependent data (DDA) on Exploris 480 to demonstrate the sensitivity of the In-insert protocol sensitivity and benchmark it with intersecting protocol published by Weke et al. (2022).