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(3) Controls. Vehicle controls, which are microbiomes cultured in the absence of compound treatment but
in the presence of compound vehicle, should be included. For example, when compounds are pre-dissolved
in DMSO, the same amount of DMSO should be used in the vehicle control. Positive controls of known
effects on the ex vivo human gut microbiome should also be included, such as fructooligosaccharide (FOS)
or kestose. In addition, a blank control of culture media without inoculation of microbiome should be
added to monitor potential contamination occurrences.

(4) Biological and technical replications. An adequate number of biological replicates should be included
[41]
and the sample size should be determined through power analysis . In terms of technical replicates, for
smaller-scale studies or individualized studies, we recommend that each condition is carried out with
technical triplicates. For large-scale studies that have sufficient power of biological replicates, the minimum
requirement is that technical triplicates of negative and positive controls should be performed.

(5) Quality controls for LC-MS/MS. For large-scale assays requiring a relatively long LC-MS/MS running
time (e.g. > 2 days), quality control (QC) samples are necessary to ensure quality, reproducibility, and
comparability of results across different batches. The importance of conducting QC runs also includes
ensuring the cleanliness of the samples. We strongly suggest that the QC sample should be study-specific.
For this, a mixture of a small aliquot from each sample is recommended. It is also recommended that the
researchers run the QC samples on LC-MS/MS to confirm sample quality before labeling by TMT.

(6) Enabling more analysis. As metaproteomics provides information on protein compositions, it does not
include other information such as genomic and metabolite compositions. We recommend that to enable
multi-omics analysis and other biochemical analysis of the samples, aliquots of samples should be saved at
certain steps for such purposes. For example, the microbial cell-free supernatant of the microbial cell
washing step may be taken/stored for possible metabolite analysis. At the final step of microbial cell
washing, before pelleting the microbial cells, samples may be divided into two aliquots for metaproteomics
and metagenomics, respectively. In addition, this protocol was developed for the aim of compound
screening purpose which does not directly enable deep metaproteomics analysis. We recommend that the
researchers save aliquots of protein lysate or digest for each sample and do fractionation-based deep
metaproteomics with specific samples of interest following the first-pass screening analysis.

DECLARATIONS
Authors’ contributions
Supervised the study: Figeys D
Developed the protocols: Li L, Mayne J, Zhang X, Ning Z
Performed the experiments: Li L, Mayne J, Beltran A
Performed data analysis and interpretation: Li L
Provided administrative support: Mayne J
Wrote the manuscript: Li L, Mayne J, Beltran A
All authors have revised and approved the manuscript.

Availability of data and materials
Source codes for the results section are provided as an RNotebook file, accessible at https://github.com/
northomics/RapidAIM_protocol_dataset.

Financial support and sponsorship
This work was supported by the Government of Canada through Genome Canada and the Ontario