fig1
Figure 1. Overview of the RapidAIM 2.0 protocol workflow. (A) Using an anaerobic chamber with a 37 °C incubator, individual fecal samples are cultured in the optimized medium with or without the stimuli of interest. A 96-well liquid handler is recommended for liquid handling. Sample plates are shaken on an orbital shaker at 500 rpm; (B) Cultured microbiome samples are then washed with PBS buffer using a centrifuge with a deepwell plate rotor; (C) Microbial cells are lysed in 96-well PCR plates using a cup-horn ultra-sonicator, and proteins are then purified using a double-precipitation procedure; (D) Proteins are then quantified and diluted, followed by automated digestion and desalting; (E) Desalted peptides are then labeled with TMT and each TMT11plexTM mix is then desalted again; (F) Samples are finally analyzed with LC-MS/MS, and *.RAW files are subjected to database search and data analysis; (G) Estimated timeline corresponding to an experiment of four 96-well plates (i.e., 320 samples). Filled squares indicate pause points to which samples can be stored at -20 °C until further processed (or otherwise -80 °C if stated). TMT: Tandem mass tags; LC-MS/MS: liquid chromatography - tandem mass spectrometry.
