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Li et al. Microbiome Res Rep 2024;3:26 https://dx.doi.org/10.20517/mrr.2023.57 Page 9 of 16

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
TM
by automated digestion and desalting; (E) Desalted peptides are then labeled with TMT and each TMT11plex 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.