title: In Vitro Metabolic Labeling of Intestinal Microbiota for Quantitative Metaproteomics.
abstract: Intestinal microbiota is emerging as one of the key environmental factors influencing or causing the development of numerous human diseases. Metaproteomics can provide invaluable information on the functional activities of intestinal microbiota and on host-microbe interactions as well. However, the application of metaproteomics in human microbiota studies is still largely limited, in part due to the lack of accurate quantitative intestinal metaproteomic methods. Most current metaproteomic microbiota studies are based on label-free quantification, which may suffer from variability during the separate sample processing and mass spectrometry runs. In this study, we describe a quantitative metaproteomic strategy, using in vitro stable isotopically ((15)N) labeled microbiota as a spike-in reference, to study the intestinal metaproteomes. We showed that the human microbiota were efficiently labeled (>95% (15)N enrichment) within 3 days under in vitro conditions, and accurate light-to-heavy protein/peptide ratio measurements were obtained using a high-resolution mass spectrometer and the quantitative proteomic software tool Census. We subsequently employed our approach to study the in vitro modulating effects of fructo-oligosaccharide and five different monosaccharides on the microbiota. Our methodology improves the accuracy of quantitative intestinal metaproteomics, which would promote the application of proteomics for functional studies of intestinal microbiota.
authors.affiliation: Ottawa Institute of Systems Biology and Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa , Ottawa, Ontario, Canada , K1H 8M5.
authors.forename: Xu
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Ottawa Institute of Systems Biology and Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa , Ottawa, Ontario, Canada , K1H 8M5.
authors.forename: Zhibin
authors.initials: Z
authors.lastname: Ning
authors.affiliation: Ottawa Institute of Systems Biology and Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa , Ottawa, Ontario, Canada , K1H 8M5.
authors.forename: Janice
authors.initials: J
authors.lastname: Mayne
authors.affiliation: Ottawa Institute of Systems Biology and Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa , Ottawa, Ontario, Canada , K1H 8M5.
authors.forename: Shelley A
authors.initials: SA
authors.lastname: Deeke
authors.affiliation: Ottawa Institute of Systems Biology and Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa , Ottawa, Ontario, Canada , K1H 8M5.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Li
authors.affiliation: Ottawa Institute of Systems Biology and Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa , Ottawa, Ontario, Canada , K1H 8M5.
authors.forename: Amanda E
authors.initials: AE
authors.lastname: Starr
authors.affiliation: Ottawa Institute of Systems Biology and Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa , Ottawa, Ontario, Canada , K1H 8M5.
authors.forename: Rui
authors.initials: R
authors.lastname: Chen
authors.affiliation: CHEO Inflammatory Bowel Disease Centre and Research Institute and Department of Paediatrics, University of Ottawa , Ottawa, Ontario, Canada , K1H 8M5.
authors.forename: Ruth
authors.initials: R
authors.lastname: Singleton
authors.affiliation: Ottawa Institute of Systems Biology and Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa , Ottawa, Ontario, Canada , K1H 8M5.
authors.forename: James
authors.initials: J
authors.lastname: Butcher
authors.affiliation: CHEO Inflammatory Bowel Disease Centre and Research Institute and Department of Paediatrics, University of Ottawa , Ottawa, Ontario, Canada , K1H 8M5.
authors.forename: David R
authors.initials: DR
authors.lastname: Mack
authors.affiliation: Ottawa Institute of Systems Biology and Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa , Ottawa, Ontario, Canada , K1H 8M5.
authors.forename: Alain
authors.initials: A
authors.lastname: Stintzi
authors.affiliation: Ottawa Institute of Systems Biology and Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa , Ottawa, Ontario, Canada , K1H 8M5.
authors.forename: Daniel
authors.initials: D
authors.lastname: Figeys
mesh_terms: D002851:Chromatography, High Pressure Liquid; D016208:Databases, Factual; D005643:Fucose / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000382:microbiology*; D007553:Isotope Labeling; D064307:Microbiota; D009587:Nitrogen Isotopes / Q000737:chemistry; D010455:Peptides / Q000032:analysis / Q000737:chemistry; D025341:Principal Component Analysis; D011506:Proteins / Q000032:analysis / Q000737:chemistry; D040901:Proteomics; D053719:Tandem Mass Spectrometry
chemical_list: D009587:Nitrogen Isotopes; C000615243:Nitrogen-15; D010455:Peptides; D011506:Proteins; D005643:Fucose