Protease profiling in fecal samples: a novel non-invasive diagnostic tool for gastrointestinal disorders

Fecal protease profiling represents a promising frontier in the non-invasive diagnosis of gastrointestinal disorders, particularly inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis, and irritable bowel syndrome (IBS). These conditions share overlapping symptoms but differ significantly in etiology and pathology, making accurate differentiation essential for appropriate management. This pilot study investigated protease activity in stool samples using a custom panel of fluorogenic peptide substrates across varying pH conditions to uncover disease-specific enzymatic signatures. Samples from IBD patients revealed broad protease activation involving both serine and cysteine classes, while the small IBS cohort showed a tendency toward a pattern enriched in furin-like serine proteases pattern dominated by furin-like serine proteases, especially at alkaline pH. Notably, one substrate, Ac-RSVL-AMC, showed higher activity in UC than in CD at acidic pH and moderate discriminatory ability in this pilot cohort. Inhibition assays confirmed the enzymatic contribution of furin-like proteases, and follow-up analysis in remission-phase IBD patients indicated persistent dysregulation, suggesting potential biomarker utility beyond active inflammation. The observed substrate-specific activity profiles highlight the importance of sequence context in proteolytic cleavage and underscore the complexity of protease involvement in gastrointestinal pathology. These findings support fecal protease profiling as a promising, rapid and low-cost approach that could contribute to distinguishing IBD from IBS and differentiating IBD subtypes, providing a foundation for future minimally invasive diagnostic strategies that require validation in larger cohorts.