Enhancing in-situ molecular profiling of gastric cancer for spatial proteomics through optimized digestion.

[BACKGROUND] Gastric cancer has a high incidence worldwide, affecting men more frequently than women. Treatment options remain limited in their success, primarily due to insufficient knowledge about the disease. The high degree of heterogeneity in gastric tissue can obscure molecular information, making comprehensive analysis challenging. Enzymatic digestion, a crucial step in protein analysis, is not yet sufficiently optimized for specific human tissue samples like gastric tissue, thereby limiting the analytical potential of techniques such as matrix-assisted laser desorption/ionization - mass spectrometry imaging (MALDI-MSI).

[RESULTS] We utilized archived specimens from 103 patient-derived samples across nine tissue types (brain, breast, kidney, lung, liver, pancreas, stomach, tonsil, and leiomyoma) to test different protocols aiming to improve tryptic digestion of formalin-fixed, paraffin-embedded tissues for in-situ proteomic analysis. The optimized digestion protocol improved peptide detection and spectral quality while preserving histological integrity. This protocol was employed to characterize a gastric cancer cohort (N = 97). MALDI-MSI data identified molecular signatures associated with disease progression, microsatellite instability status, treatment response, and spatial distribution.

[SIGNIFICANCE] We gained access to molecular information on tumor progression and treatment response by employing a tailored tryptic digestion protocol for MALDI-MSI detection. By advancing molecular profiling in gastric cancer, these findings provide valuable insight into disease-related molecular changes and contribute to developing more precise and personalized diagnostic strategies.