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A Method for Comprehensive Proteomic Analysis of Human Faecal Samples to Investigate Gut Dysbiosis in Patients with Cystic Fibrosis

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Emerging Sample Treatments in Proteomics

Part of the book series: Advances in Experimental Medicine and Biology ((PMISB,volume 1073))

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.

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Notes

  1. 1.

    Since 2013, the database has been dramatically increased, e.g. by inclusion of several metagenomics projects. Therefore, the number of identified peptides/proteins could probably be improved by researching data against recent updates of the database. However, the aim of this paper is to compare protocols, and the main conclusions do not depend on the database used.

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Acknowledgements

This research was supported by grant G.0638.10 from Research Foundation Flanders (FWO). PD acknowledges the support of Ghent University (MRP Bioinformatics: from nucleotides to networks). The authors thank Dr. Kris Moreel for the generous help with the LC-MS/MS analyses on the FT-ICR-MS.

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Authors and Affiliations

  1. Department of Biochemistry and Microbiology, Ghent University, Ghent, Belgium

    Griet Debyser, Maarten Aerts, Pieter Van Hecke, Gwen Duytschaever, Peter Vandamme & Bart Devreese

  2. Department of Applied Mathematics, Computer Science and Statistics, Ghent University, Ghent, Belgium

    Bart Mesuere & Peter Dawyndt

  3. Department of Pediatrics, University Hospital of Leuven, Leuven, Belgium

    Kris De Boeck

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  1. Griet Debyser
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Correspondence to Bart Devreese .

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  1. BIOSCOPE Group, LAQV-REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Proteomass Scientific Society, Rua dos Inventores, Madan Park, Caparica, Portugal

    José-Luis Capelo-Martínez

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Debyser, G. et al. (2019). A Method for Comprehensive Proteomic Analysis of Human Faecal Samples to Investigate Gut Dysbiosis in Patients with Cystic Fibrosis. In: Capelo-Martínez, JL. (eds) Emerging Sample Treatments in Proteomics. Advances in Experimental Medicine and Biology(), vol 1073. Springer, Cham. https://doi.org/10.1007/978-3-030-12298-0_6

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