Document resource
Introduction Dysregulated proteolytic activity contributes to the pathophysiology of IBD, and measurement of protease dynamics has therefore been suggested as a potential diagnostic biomarker or treatment target. Studies have, however, generally focussed on expression profiles rather than enzyme activity, with limited reference to IBD activity. This study aimed to characterise protease activity in an inception cohort of IBD patients compared to healthy controls (HC).Methods Sixty patients with IBD (30 with Crohn’s disease, 30 with Ulcerative Colitis) and 20 matched HC were recruited and faecal samples collected. All IBD patients were recruited prior to starting treatment. Following faecal protein extraction, protease activity was assessed using fluorogenic substrates selective for serine proteases. LC-MS/MS whole proteomics and chemoproteomics with activity-based probe (ABP) labelling were also performed. A biotinylated fluorophosphonate probe was used for labelling which binds to serine proteases in an activity dependent manner, allowing for the identification of active proteases. ABP samples were run with dimethyl sulfoxide (DMSO) incubated controls. All LC-MS/MS was performed following trypsin (Sigma-Aldrich) digest. Raw proteomic data were analysed using Fragpipe (v22.0) and were searched against a human protein database (Uniprot). Further proteomic and statistical analysis was performed using Perseus (v2.0.11) and GraphPad Prism (v10.6.1). Functional classification of proteins was performed using the PANTHER tool.Results Substrate assays showed significantly higher activity levels in IBD patients relative to HC when assessing chymotrypsin-like (CA), elastase-like (EA) and trypsin-like (TA) activity (p=0.0264, p=0.0236, p=0.0293 respectively; figure 1). Levels were also statistically significantly higher across all substrates in IBD patients with increased gastrointestinal inflammation indicated by raised faecal calprotectin levels (>150µg/g)(CA p=0.0016, EA p=0.0034, TA p=0.04). Whole proteomics revealed 55 statistically significantly elevated proteins in IBD patients compared to HC (FDR of 0.05 and S0 of 0.1); further analysis using PANTHER revealed 34% of these to be related to catalytic activity. ABP labelled samples demonstrated enrichment of serine hydrolases when comparing IBD to HC, and to DMSO control samples, including CELA2A which is known to exert both elastase and chymotrypsin like activityConclusions These results demonstrate increased levels of protease activity in IBD patients relative to HC. They also show higher levels in IBD patients with greater disease activity, which has not previously been well described. ABP labelling was able to identify specific enzymes driving this difference, enabling the identification of possible future biomarkers.Abstract P195 Figure 1Protease activity compared between IBD patients and healthy controls. Rate of cleavage expressed in change of relative fluorescent units per second per mg of protein