title: Development of an Integrated Pipeline for Profiling Microbial Proteins from Mouse Fecal Samples by LC-MS/MS.
abstract: Metaproteomics is one approach to analyze the functional capacity of the gut microbiome but is limited by the ability to evenly extract proteins from diverse organisms within the gut. Herein, we have developed a pipeline to optimize sample preparation of stool obtained from germ-free (GF) mice that were gavaged a defined community of 11 bacterial strains isolated from the human gut. With 64% more proteins identified, bead-beating was confirmed to be an indispensable step for the extraction of bacterial proteins, especially for Gram-positive bacteria. Bacterial enrichment from mouse fecal samples was further optimized by evaluating three different methods: (1) a high-speed differential centrifugation (HCE) or (2) a low-speed differential centrifugation (LCE) and (3) a filter-aided method (FA). The HCE method was associated with dramatic loss of bacteria and 71% less recovery of bacterial proteins than the LCE method. Compared with LCE, the FA method also showed dramatic loss of the amount of bacteria recovered and decreased protein identifications from Gram-positive bacteria in the stool samples. Ultimately, LCE may provide an alternative and complementary method for enriching bacteria from small amounts of mouse fecal samples, which could aid in investigating bacterial function in health and disease.
authors: Department of Surgery, University of Michigan , Ann Arbor, Michigan 48109, United States.
authors: Jing
authors: J
authors: Wu
authors: Department of Surgery, University of Michigan , Ann Arbor, Michigan 48109, United States.
authors: Jianhui
authors: J
authors: Zhu
authors: Department of Surgery, University of Michigan , Ann Arbor, Michigan 48109, United States.
authors: Haidi
authors: H
authors: Yin
authors: Department of Surgery, University of Michigan , Ann Arbor, Michigan 48109, United States.|Experiment Center for Science and Technology, Shanghai University of Traditional Chinese Medicine , Shanghai 201203, China.
authors: Xinhua
authors: X
authors: Liu
authors: Department of Surgery, University of Michigan , Ann Arbor, Michigan 48109, United States.
authors: Mingrui
authors: M
authors: An
authors: Department of Microbiology and Immunology, University of Michigan , Ann Arbor, Michigan 48109, United States.
authors: Nicholas A
authors: NA
authors: Pudlo
authors: Department of Microbiology and Immunology, University of Michigan , Ann Arbor, Michigan 48109, United States.
authors: Eric C
authors: EC
authors: Martens
authors: Division of Hematology/Oncology, Department of Internal Medicine, University of Michigan , Ann Arbor, Michigan 48109, United States.
authors: Grace Y
authors: GY
authors: Chen
authors: Department of Surgery, University of Michigan , Ann Arbor, Michigan 48109, United States.
authors: David M
authors: DM
authors: Lubman
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000302:isolation & purification*; D002499:Centrifugation, Density Gradient; D002623:Chemistry Techniques, Analytical / Q000379:methods* / Q000592:standards; D005243:Feces / Q000382:microbiology; D005374:Filtration; D000069196:Gastrointestinal Microbiome; D006094:Gram-Positive Bacteria / Q000737:chemistry; D006801:Humans; D051379:Mice
chemical_list: D001426:Bacterial Proteins
keywords: LC−MS/MS; bead-beating; metaproteome; microbial enrichment; protein extraction