Sample Processing for Metaproteomic Analysis of Human Gut Microbiota

Human gut microbiota can be studied through the characterization of microorganisms present in feces. Metaproteomics has arisen as a good approach to investigate this vast community. However, the processing of fecal samples in order to obtain the largest number of proteins from gut microbiota to be subsequently analyzed by means of metaproteomics is a challenge. Here we describe a protocol to approach this task. It includes two main steps: the first step of humectation and dispersion of the feces, followed by the separation of microorganisms from other fecal components such as roughage and food debris, and the second step in which microbial cells are broken up and microbiota proteins recovered for MS analysis. Detailed procedures for sample preparation, protein extraction, trypsin digestion, and mass spectrometry analysis for gut microbiota samples are provided.