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

Peter S Thuy-Boun,Ana Y Wang,Ana Crissien-Martinez,Janice H Xu,Sandip Chatterjee,Gregory S Stupp,Andrew I Su,Walter J Coyle,Dennis W Wolan

doi:10.1016/j.mcpro.2022.100197

Abstract

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.

Highlights

Inflammatory bowel disease (IBD) is a chronic medical condition characterized by relapsing inflammation of the gastrointestinal (GI) tract
Using a pipeline that incorporates a novel, strong-acid sample preparation procedure [50], label-free high resolution LC-MS/MS, and the ComPIL database coupled to the ProLuCID/SEQUEST search engine [20,51,52], we identify 176 protein groups and several gene ontology (GO) terms enriched in either cohort [53]
By LC-MS/MS proteomics, we identified 46 phyla, including Ascomycota, Basidiomycota, Spirochaetes, Chordata, and Streptophya in addition to the 8 identified by 16S rRNA gene sequencing alone (Euryarchaeota, Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, Fusobacteria, Proteobacteria and Verrucomicrobia) (Fig. 1)

Summary

Introduction

Inflammatory bowel disease (IBD) is a chronic medical condition characterized by relapsing inflammation of the gastrointestinal (GI) tract. This disease is broadly divisible into two categories based on where inflammation occurs. The incidence and prevalence of IBD in developed countries has steadily increased in the last few decades, making this disease a public health concern with a potentially heavy cost burden due to a requirement for long-term management [4]. Genome wide association studies (GWAS) have identified over 200 genetic loci associated with UC and CD, but the polygenic nature of these conditions explains only a minor portion of disease incidence [5,6,7]. IBD triggers are unknown, but its progression is hypothesized to be amplified by inappropriate host-microbe interactions that lead to dysbiosis and, eventually, observable gross pathology [9,10,11,12,13]

Objectives

Results

Conclusion