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

  • Griet Debyser
  • Maarten Aerts
  • Pieter Van Hecke
  • Bart Mesuere
  • Gwen Duytschaever
  • Peter Dawyndt
  • Kris De Boeck
  • Peter Vandamme
  • Bart DevreeseEmail author
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1073)


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.



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.


  1. 1.
    Dethlefsen L, Eckburg PB, Bik EM et al (2006) Assembly of the human intestinal microbiota. Trends Ecol Evol 21:517–523CrossRefGoogle Scholar
  2. 2.
    Gill SR, Pop M, DeBoy RT et al (2006) Metagenomic analysis of the human distal gut microbiome. Science 312:1355–1359CrossRefGoogle Scholar
  3. 3.
    Cummings JH, Macfarlane GT (1997) Role of intestinal bacteria in nutrient metabolism. JPEN J Parenter Enteral Nutr 21:357–365CrossRefGoogle Scholar
  4. 4.
    Guarner F, Malagelada JR (2003) Gut flora in health and disease. Lancet 361:512–519CrossRefGoogle Scholar
  5. 5.
    Sekirov I, Russell SL, Antunes LC et al (2010) Gut microbiota in health and disease. Physiol Rev 90:859–904CrossRefGoogle Scholar
  6. 6.
    Pryde SE, Duncan SH, Hold GL et al (2002) The microbiology of butyrate formation in the human colon. FEMS Microbiol Lett 217:133–139CrossRefGoogle Scholar
  7. 7.
    Ubeda C, Pamer EG (2012) Antibiotics, microbiota, and immune defense. Trends Immunol 33:459CrossRefGoogle Scholar
  8. 8.
    Mortensen PB, Clausen MR (1996) Short-chain fatty acids in the human colon: relation to gastrointestinal health and disease. Scand J Gastroenterol Suppl 216:132–148CrossRefGoogle Scholar
  9. 9.
    Barcenilla A, Pryde SE, Martin JC et al (2000) Phylogenetic relationships of butyrate-producing bacteria from the human gut. Appl Environ Microbiol 66:1654–1661CrossRefGoogle Scholar
  10. 10.
    Duytschaever G, Huys G, Bekaert M et al (2013) Dysbiosis of bifidobacteria and Clostridium cluster XIVa in the cystic fibrosis fecal microbiota. J Cyst Fibros 12:206–215CrossRefGoogle Scholar
  11. 11.
    Tannock GW (2008) The search for disease-associated compositional shifts in bowel bacterial communities of humans. Trends Microbiol 16:488–495CrossRefGoogle Scholar
  12. 12.
    Larsen N, Vogensen FK, van den Berg FW et al (2010) Gut microbiota in human adults with type 2 diabetes differs from non-diabetic adults. PLoS One 5:e9085CrossRefGoogle Scholar
  13. 13.
    van Tongeren SP, Slaets JP, Harmsen HJ et al (2005) Fecal microbiota composition and frailty. Appl Environ Microbiol 71:6438–6442CrossRefGoogle Scholar
  14. 14.
    Balamurugan R, Rajendiran E, George S et al (2008) Real-time polymerase chain reaction quantification of specific butyrate-producing bacteria, Desulfovibrio and Enterococcus faecalis in the feces of patients with colorectal cancer. J Gastroenterol Hepatol 23:1298–1303CrossRefGoogle Scholar
  15. 15.
    Davies JC, Bilton D (2009) Bugs, biofilms, and resistance in cystic fibrosis. Respir Care 54:628–640CrossRefGoogle Scholar
  16. 16.
    Wilschanski M, Durie PR (2007) Patterns of GI disease in adulthood associated with mutations in the CFTR gene. Gut 56:1153–1163CrossRefGoogle Scholar
  17. 17.
    O’Brien S, Mulcahy H, Fenlon H et al (1993) Intestinal bile acid malabsorption in cystic fibrosis. Gut 34:1137–1141CrossRefGoogle Scholar
  18. 18.
    Duytschaever G, Huys G, Bekaert M et al (2011) Cross-sectional and longitudinal comparisons of the predominant fecal microbiota compositions of a group of pediatric patients with cystic fibrosis and their healthy siblings. Appl Environ Microbiol 77:8015–8024CrossRefGoogle Scholar
  19. 19.
    Bruzzese E, Raia V, Gaudiello G et al (2004) Intestinal inflammation is a frequent feature of cystic fibrosis and is reduced by probiotic administration. Aliment Pharmacol Ther 20:813–819CrossRefGoogle Scholar
  20. 20.
    Hawrelak JA, Myers SP (2004) The causes of intestinal dysbiosis: a review. Altern Med Rev 9:180–197PubMedGoogle Scholar
  21. 21.
    Modolell I, Guarner L, Malagelada JR (2002) Digestive system involvement in cystic fibrosis. Pancreatology 2:12–16CrossRefGoogle Scholar
  22. 22.
    Verberkmoes NC, Russell AL, Shah M et al (2009) Shotgun metaproteomics of the human distal gut microbiota. ISME J 3:179–189CrossRefGoogle Scholar
  23. 23.
    Rooijers K, Kolmeder C, Juste C et al (2011) An iterative workflow for mining the human intestinal metaproteome. BMC Genomics 12:6CrossRefGoogle Scholar
  24. 24.
    Kolmeder CA, de Been M, Nikkila J et al (2012) Comparative metaproteomics and diversity analysis of human intestinal microbiota testifies for its temporal stability and expression of core functions. PLoS One 7:e29913CrossRefGoogle Scholar
  25. 25.
    Keller A, Nesvizhskii AI, Kolker E et al (2002) Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem 74:5383–5392CrossRefGoogle Scholar
  26. 26.
    Nesvizhskii AI, Keller A, Kolker E et al (2003) A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem 75:4646–4658CrossRefGoogle Scholar
  27. 27.
    Mesuere B, Devreese B, Debyser G et al (2012) Unipept: tryptic peptide-based biodiversity analysis of metaproteome samples. J Proteome Res 11:5773–5780CrossRefGoogle Scholar
  28. 28.
    Conesa A, Gotz S, Garcia-Gomez JM et al (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676CrossRefGoogle Scholar
  29. 29.
    Quevillon E, Silventoinen V, Pillai S et al (2005) InterProScan: protein domains identifier. Nucleic Acids Res 33:W116–W120CrossRefGoogle Scholar
  30. 30.
    Myhre S, Tveit H, Mollestad T et al (2006) Additional gene ontology structure for improved biological reasoning. Bioinformatics 22:2020–2027CrossRefGoogle Scholar
  31. 31.
    Kanehisa M, Goto S, Sato Y et al (2012) KEGG for integration and interpretation of large-scale molecular data sets. Nucleic Acids Res 40:D109–D114CrossRefGoogle Scholar
  32. 32.
    Detlefsen L, McFall-Ngai M, Relman DA (2007) An ecological and evolutionary perspective on human-microbe mutualism and disease. Nature 449:811–818CrossRefGoogle Scholar
  33. 33.
    Liu C, Finegold SM, Song Y et al (2008) Reclassification of Clostridium coccoides, Ruminococcus hansenii, Ruminococcus hydrogenotrophicus, Ruminococcus luti, Ruminococcus productus and Ruminococcus schinkii as Blautia coccoides gen. nov., comb. nov., Blautia hansenii comb. nov., Blautia hydrogenotrophica comb. nov., Blautia luti comb. nov., Blautia producta comb. nov., Blautia schinkii comb. nov. and description of Blautia wexlerae sp. nov., isolated from human faeces. Int J Syst Evol Microbiol 58:1896–1902CrossRefGoogle Scholar
  34. 34.
    Gerritsen J, Smidt H, Rijkers GT et al (2011) Intestinal microbiota in human health and disease: the impact of probiotics. Genes Nutr 6:209–240CrossRefGoogle Scholar
  35. 35.
    Ben-Amor K, Heilig H, Smidt H et al (2005) Genetic diversity of viable, injured, and dead fecal bacteria assessed by fluorescence-activated cell sorting and 16S rRNA gene analysis. Appl Environ Microbiol 71:4679–4689CrossRefGoogle Scholar
  36. 36.
    Bahl MI, Bergstrom A, Licht TR (2012) Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis. FEMS Microbiol Lett 329:193–197CrossRefGoogle Scholar
  37. 37.
    Neilson KA, Ali NA, Muralidharan S et al (2011) Less label, more free: approaches in label-free quantitative mass spectrometry. Proteomics 11:535–553CrossRefGoogle Scholar
  38. 38.
    Okazaki N, Takahashi N, Kojima S et al (2002) Protocadherin LKC, a new candidate for a tumor suppressor of colon and liver cancers, its association with contact inhibition of cell proliferation. Carcinogenesis 23:1139–1148CrossRefGoogle Scholar
  39. 39.
    Apajalahti JH, Sarkilahti LK, Maki BR et al (1998) Effective recovery of bacterial DNA and percent-guanine-plus-cytosine-based analysis of community structure in the gastrointestinal tract of broiler chickens. Appl Environ Microbiol 64:4084–4088PubMedPubMedCentralGoogle Scholar
  40. 40.
    Kolmeder CA, de Vos WM (2014) Metaproteomics of our microbiome - developing insight in function and activity in man and model systems. J Proteome 97:3–16CrossRefGoogle Scholar
  41. 41.
    Ham BM, Yang F, Jayachandran H et al (2008) The influence of sample preparation and replicate analyses on HeLa cell phosphoproteome coverage. J Proteome Res 7:2215–2221CrossRefGoogle Scholar
  42. 42.
    Zhang X, Chen W, Ning Z et al (2017) Deep Metaproteomics approach for the study of human microbiomes. Anal Chem 89:9407–9415CrossRefGoogle Scholar
  43. 43.
    Lozupone CA, Stombaugh JI, Gordon JI et al (2012) Diversity, stability and resilience of the human gut microbiota. Nature 489:220–230CrossRefGoogle Scholar
  44. 44.
    Duytschaever G, Huys G, Boulanger L et al (2013) Amoxicillin-clavulanic acid resistance in fecal Enterobacteriaceae from patients with cystic fibrosis and healthy siblings. J Cyst Fibros 12:780CrossRefGoogle Scholar
  45. 45.
    Schippa S, Iebba V, Santangelo F et al (2013) Cystic fibrosis transmembrane conductance regulator (CFTR) allelic variants relate to shifts in faecal microbiota of cystic fibrosis patients. PLoS One 8:e61176CrossRefGoogle Scholar
  46. 46.
    Del Campo R, Garriga M, Perez-Aragon A et al (2014) Improvement of digestive health and reduction in proteobacterial populations in the gut microbiota of cystic fibrosis patients using a Lactobacillus reuteri probiotic preparation: a double blind prospective study. J Cyst Fibros 13:716–722Google Scholar
  47. 47.
    Sokol H, Seksik P, Furet JP et al (2009) Low counts of Faecalibacterium prausnitzii in colitis microbiota. Inflamm Bowel Dis 15:1183–1189CrossRefGoogle Scholar
  48. 48.
    Willing BP, Dicksved J, Halfvarson J et al (2010) A pyrosequencing study in twins shows that gastrointestinal microbial profiles vary with inflammatory bowel disease phenotypes. Gastroenterology 139:1844–1854 e1841CrossRefGoogle Scholar
  49. 49.
    Debyser G, Mesuere B, Clement L et al (2016) Faecal proteomics: a tool to investigate dysbiosis and inflammation in patients with cystic fibrosis. J Cyst Fibros 15:242–250CrossRefGoogle Scholar
  50. 50.
    Ladirat SE, Schols HA, Nauta A et al (2013) High-throughput analysis of the impact of antibiotics on the human intestinal microbiota composition. J Microbiol Methods 92:387–397CrossRefGoogle Scholar
  51. 51.
    Jernberg C, Lofmark S, Edlund C et al (2007) Long-term ecological impacts of antibiotic administration on the human intestinal microbiota. ISME J 1:56–66CrossRefGoogle Scholar
  52. 52.
    Jakobsson HE, Jernberg C, Andersson AF et al (2010) Short-term antibiotic treatment has differing long-term impacts on the human throat and gut microbiome. PLoS One 5:e9836CrossRefGoogle Scholar
  53. 53.
    Perez-Cobas AE, Gosalbes MJ, Friedrichs A et al (2013) Gut microbiota disturbance during antibiotic therapy: a multi-omic approach. Gut 62:1591–1601CrossRefGoogle Scholar
  54. 54.
    Turroni F, Peano C, Pass DA et al (2012) Diversity of Bifidobacteria within the infant gut microbiota. PLoS One 7:e36957CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Griet Debyser
    • 1
  • Maarten Aerts
    • 1
  • Pieter Van Hecke
    • 1
  • Bart Mesuere
    • 2
  • Gwen Duytschaever
    • 1
  • Peter Dawyndt
    • 2
  • Kris De Boeck
    • 3
  • Peter Vandamme
    • 1
  • Bart Devreese
    • 1
    Email author
  1. 1.Department of Biochemistry and MicrobiologyGhent UniversityGhentBelgium
  2. 2.Department of Applied Mathematics, Computer Science and StatisticsGhent UniversityGhentBelgium
  3. 3.Department of PediatricsUniversity Hospital of LeuvenLeuvenBelgium

Personalised recommendations