Mapping the Spatial Distribution of Tryptic Peptides in Formalin-Fixed Paraffin-Embedded Tissues Using Desorption Electrospray Ionization Mass Spectrometry Imaging

Spatial proteomics visualizes protein localization within native cellular and tissue environments, providing critical insights into pathological processes, tissue heterogeneity, and diagnostic biomarkers. Mass spectrometry imaging (MSI) has emerged as a powerful tool for spatial proteomics, with matrix-assisted laser desorption/ionization (MALDI)-MSI being the most widely used platform. Recently, desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) has been explored for mapping proteins and peptides in tissue sections, with distinct sets of proteins detected compared to MALDI MSI. However, current studies have focused on fresh-frozen tissues, and the application of DESI-MSI to formalin-fixed paraffin-embedded (FFPE) tissues, the predominant form of clinical specimens, remains unexplored. Here, we report the optimization and application of DESI-MSI for proteomic analysis of FFPE tissues. Sample pretreatment procedures and DESI parameters were systematically optimized using FFPE mouse brain tissue sections to enhance tryptic peptide detection. The optimized workflow was further applied to FFPE canine melanocytoma tissue to discover protein biomarkers. Protein identification was performed using nanoLC-MS/MS. Overall, we demonstrate the potential of DESI-MSI as a platform for spatial proteomic analysis of FFPE tissues, expanding its applicability to archived clinical specimens.