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Page 8 of 16 Li et al. Microbiome Res Rep 2024;3:26 https://dx.doi.org/10.20517/mrr.2023.57

component analysis and hierarchical clustering using RMarkdown.

Timing of each step:
Microbiome culturing and treatment, 1-2 days;
Microbial cell washing, 5-6 h;
Microbial cell lysis and protein double-precipitation, 2 days;
Protein digestion, 1 day;
Desalting, 1-2 h;
TMT-labeling and desalting, 4-5 h;
LC-MS/MS analysis, 2.5 h per TMT11plex.

RESULTS
Development of the RapidAIM 2.0 approach
The RapidAIM 2.0 approach is an updated version of the previous RapidAIM approaches developed and
used in research studies by our laboratory [18,35] . In the previous workflows, we described a 96-well-based
workflow to study the functional responses of individual gut microbiomes to xenobiotics in vitro. The
development of the previous RapidAIM method consisted of three major stages, (1) optimized the culture
medium and established and validated the 96-well-based scalable culturing model [19,36] ; (2) established a 96-
[18]
well-based metaproteomic sample processing and data analysis workflow ; (3) and most recently, we
developed and validated a live microbiota biobanking workflow that is helpful to increase the
[35]
reproducibility of experiments . A limitation of this previous protocol was the considerably large sample
size and LC-MS/MS time consumption for metaproteomics analysis. Therefore, we updated the protocol to
overcome this limitation. New features of the protocol include (1) optimization of the protein extraction
and purification protocol; (2) automation of the protein digestion and desalting protocol; (3) introduction
of TMT multiplexing technique for labeling and quantitation of peptides and proteins, allowing for the
analysis of up to 10 samples in one LC-MS/MS run ; and (4) a TMT-based statistical analysis streamline
[34]
for clustering functional responses. We estimate that, for an experiment containing 320 samples (four 96-
well plates), this updated workflow requires only six days for culturing and sample processing, and it
shortens LC-MS/MS sample analysis time from approximately 20 to 3 days.

As illustrated in Figure 1, the RapidAIM 2.0 protocol is divided into six sequential stages: (A) microbiome
collection, culturing, and compound treatment; (B) microbial cell washing; (C) protein extraction and
purification; (D) protein digestion and desalting; (E) TMT labeling and desalting; (F) LC-MS/MS analysis
and metaproteomic data analysis. First, individual fecal samples are cultured with or without compounds/
stimuli of interest [Figure 1A]. An anaerobic chamber with a 37 °C incubator capable of accommodating an
orbital shaker is used. A 96-well liquid handler is recommended for liquid handling. Sample plates are
shaken on an orbital shaker at 500 rpm for 18-48 h. Either fresh human fecal samples or -80 °C stored
biobank samples can be used for the culturing step. We previously showed that our culturing protocol
maintains the functionality of individual microbiomes with both sample types [18,35] .

Supplementary Methods 1 and 2 provide details for collecting and processing fresh stool samples, and
collecting, processing and biobanking of the samples, respectively.

Next, microbial cells are purified using differential centrifugation and then stored at -80 °C [Figure 1B].
Proteins are then extracted from the cells and purified using a double-precipitation procedure [Figure 1C].
After protein quantification, samples are diluted to a recommended protein concentration of 1 µg/µL,
followed by automated digestion and desalting procedures [Figure 1D and Supplementary Figure 1A and B].