ABSTRACT
BACKGROUND: This chapter reports the evaluation of two shotgun metaproteomic workflows. The methods were developed to investigate gut dysbiosis via analysis of the faecal microbiota from patients with cystic fibrosis (CF). We aimed to set up an unbiased and effective method to extract the entire proteome, i.e. to extract sufficient bacterial proteins from the faecal samples in combination with a maximum of host proteins giving information on the disease state. METHODS: Two protocols were compared; the first method involves an enrichment of the bacterial proteins while the second method is a more direct method to generate a whole faecal proteome extract. The different extracts were analysed using denaturing polyacrylamide gel electrophoresis followed by liquid chromatography-tandem mass spectrometry aiming a maximal coverage of the bacterial protein content in faecal samples. RESULTS AND CONCLUSIONS: In all extracts, microbial proteins are detected, and in addition, nonbacterial proteins are detected in all samples providing information about the host status. Our study demonstrates the huge influence of the used protein extraction method on the obtained result and shows the need for a standardised and appropriate sample preparation for metaproteomic analysis. To address questions on the health status of the patients, a whole protein extract is preferred over a method to enrich the bacterial fraction. In addition, the method of the whole protein fraction is faster, which gives the possibility to analyse more biological replicates.
Subject(s)
Cystic Fibrosis/complications , Dysbiosis/diagnosis , Feces/chemistry , Proteome , Proteomics/methods , Bacterial Proteins/analysis , Chromatography, Liquid , Humans , Tandem Mass SpectrometryABSTRACT
BACKGROUND: Several microbial studies reported gut microbiota dysbiosis in patients with cystic fibrosis (CF). The functional consequences of this phenomenon are poorly understood. Faecal metaproteomics allows the quantitative analysis of host and microbial proteins to address functional changes resulting from this dysbiosis. METHODS: We analysed faecal protein extracts from fifteen patients with CF that have pancreatic insufficiency and from their unaffected siblings by shotgun proteomics. Novel computational and statistical tools were introduced to evaluate changes in taxonomic composition and protein abundance. RESULTS: Faecal protein extracts from patients with CF were dominated by host proteins involved in inflammation and mucus formation. Taxonomic analysis of the microbial proteins confirmed the strong reduction of butyrate reducers such as Faecalibacterium prausnitzii and increase of Enterobacteriaceae, Ruminococcus gnavus and Clostridia species. CONCLUSION: Faecal metaproteomics provides insights in intestinal dysbiosis, inflammation in patients with CF and can be used to monitor different disease markers in parallel.
