Novel protocol for spatial proteome analysis of 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). In-insert proteomics represents a blueprint in spatial FFPE tissue subsection processing by completely omitting the robotic platform, desalting prior to injection to the liquid chromatograph and detergent removal while restraining protein losses to the minimum. In-insert proteomics has a sufficient sensitivity to preserve spatial context within a single FFPE tissue slide. Spatial pathway regulation trends were evaluated between 17 individual breast tissue voxels by the method. Bioinformatic spatial analysis of the most dysregulated proteins was performed to reveal hotspots of certain biochemical signaling/processes. A special attention was given to cytoskeleton reorganization as an effect of hormone signaling.