Refining Spatial Proteomics by Mass Spectrometry: An Efficient Workflow Tailored for Archival Tissue

Formalin-fixed paraffin-embedded (FFPE) tissue, while excellent for preserving tissue for extended periods of time, poses a challenge when extracting molecular information. We therefore developed an easily adaptable and highly efficient workflow for extracting high levels of proteins from low-input material. We compared sensitivity between two stains, EpCAM and H&E, across material inputs of 1,166 and ∼800,000 µ m2. In the context of EpCAM-stained tissue, our investigations unveiled a range from ∼1,200 unique protein groups at the lowest input to ∼5,900 at the highest. For H&E, the spectrum covers ∼900 to ∼5,200 protein groups. We found an optimal balance between maximizing detected proteins and minimizing input material to be within the range of ∼50,000 to ∼100,000 µ m2. With this knowledge, we tested the spatial capabilities by isolating specific cell populations, through Laser Capture Microdissection (LCM), from three different tissue types, where we were able to identify tissue-specific signatures and prominent clustering of all cell populations. ### Competing Interest Statement The authors have declared no competing interest.