Quantitative Metaproteomics and Activity-based Protein Profiling of Patient Fecal Microbiome Identifies Host and Microbial Serine-type Endopeptidase Activity Associated With Ulcerative Colitis

• Published in: Molecular & cellular proteomics 2022-03, Vol.21 (3), p.100197-100197, Article 100197
• Main Authors: Thuy-Boun, Peter S., Wang, Ana Y., Crissien-Martinez, Ana, Xu, Janice H., Chatterjee, Sandip, Stupp, Gregory S., Su, Andrew I., Coyle, Walter J., Wolan, Dennis W.
• Format: Article
• Language: English
• Subjects: ABPP; activity-based protein profiling; chemoproteomics; Chromatography, Liquid; Colitis, Ulcerative - diagnosis; Colitis, Ulcerative - microbiology; ComPIL; de novo peptide sequencing; Endopeptidases; Feces - microbiology; fluorophosphonate; Humans; microbiome metaproteomics; Microbiota; RNA, Ribosomal, 16S - genetics; Serine; Tandem Mass Spectrometry; ulcerative colitis
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1016/j.mcpro.2022.100197

The gut microbiota plays an important yet incompletely understood role in the induction and propagation of ulcerative colitis (UC). Organism-level efforts to identify UC-associated microbes have revealed the importance of community structure, but less is known about the molecular effectors of disease. We performed 16S rRNA gene sequencing in parallel with label-free data-dependent LC-MS/MS proteomics to characterize the stool microbiomes of healthy (n = 8) and UC (n = 10) patients. Comparisons of taxonomic composition between techniques revealed major differences in community structure partially attributable to the additional detection of host, fungal, viral, and food peptides by metaproteomics. Differential expression analysis of metaproteomic data identified 176 significantly enriched protein groups between healthy and UC patients. Gene ontology analysis revealed several enriched functions with serine-type endopeptidase activity overrepresented in UC patients. Using a biotinylated fluorophosphonate probe and streptavidin-based enrichment, we show that serine endopeptidases are active in patient fecal samples and that additional putative serine hydrolases are detectable by this approach compared with unenriched profiling. Finally, as metaproteomic databases expand, they are expected to asymptotically approach completeness. Using ComPIL and de novo peptide sequencing, we estimate the size of the probable peptide space unidentified (“dark peptidome”) by our large database approach to establish a rough benchmark for database sufficiency. Despite high variability inherent in patient samples, our analysis yielded a catalog of differentially enriched proteins between healthy and UC fecal proteomes. This catalog provides a clinically relevant jumping-off point for further molecular-level studies aimed at identifying the microbial underpinnings of UC. [Display omitted] •Identified 176 significantly altered protein groups between healthy and UC patients.•Serine-type endopeptidase activity is overrepresented in UC patients.•Fluorophosphonate ABPP shows that endopeptidases are active in fecal samples.•ABPP enrichment helps identify additional putative serine hydrolases in samples.•De novo sequencing used to estimate number of MS2 spectra unidentified by ComPIL. Thuy-Boun et al. quantitatively compare the stool microbiomes of healthy and ulcerative colitis patients with label-free data-dependent LC-MS/MS proteomics. Their analyses identified 176 significantly enriche