Stool Processing for Functional Characterization by Activity-Based Protein Profiling of Bacterial Enzymes in Complex Human Gut Microbiome Systems

The human gut microbiome is a complex ecosystem composed of trillions of microorganisms whose metabolic activities influence human health, disease progression, and therapeutic outcomes. While sequencing-based approaches provide valuable information regarding microbial composition and genetic potential, they do not directly measure enzyme activity within microbial communities. Activity-based protein profiling (ABPP) enables the selective labeling, enrichment, and identification of active enzymes, providing a functional readout of microbiome activity. Successful application of ABPP to human stool samples requires robust sample processing methods that efficiently isolate bacterial cells while minimizing interference from dietary material, host-derived components, and other contaminants present within the fecal matrix. The goal of this protocol is to provide a reproducible workflow for processing human stool samples for functional characterization of active bacterial enzymes using ABPP and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics. This method describes stool homogenization, bacterial cell isolation, lysate preparation, probe labeling, enrichment of labeled proteins, and downstream LC-MS/MS measurement. The protocol is designed to generate high-quality bacterial protein extracts suitable for profiling active enzyme populations within complex gut microbiome samples.