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ToolsLi, Leyuan, Mayne, Janice, Beltran, Adrian, Zhang, Xu, Ning, Zhibin and Figeys, Daniel (2024) RapidAIM 2.0: a high-throughput assay to study functional response of human gut microbiome to xenobiotics. Microbiome Research Reports, 3 (2). ISSN 2771-5965
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Abstract
Aim: Our gut microbiome has its own functionalities which can be modulated by various xenobiotic and biotic components. The development and application of a high-throughput functional screening approach of individual gut microbiomes accelerates drug discovery and our understanding of microbiome-drug interactions. We previously developed the rapid assay of individual microbiome (RapidAIM), which combined an optimized culturing model with metaproteomics to study gut microbiome responses to xenobiotics. In this study, we aim to incorporate automation and multiplexing techniques into RapidAIM to develop a high-throughput protocol. Methods: To develop a 2.0 version of RapidAIM, we automated the protein analysis protocol, and introduced a tandem mass tag (TMT) multiplexing technique. To demonstrate the typical outcome of the protocol, we used RapidAIM 2.0 to evaluate the effect of prebiotic kestose on ex vivo individual human gut microbiomes biobanked with five different workflows. Results: We describe the protocol of RapidAIM 2.0 with extensive details on stool sample collection, biobanking, in vitro culturing and stimulation, sample processing, metaproteomics measurement, and data analysis. The analysis depth of 5,014 +/- 142 protein groups per multiplexed sample was achieved. A test on five biobanking methods using RapidAIM 2.0 showed the minimal effect of sample processing on live microbiota functional responses to kestose. Conclusions: Depth and reproducibility of RapidAIM 2.0 are comparable to previous manual label-free metaproteomic analyses. In the meantime, the protocol realizes culturing and sample preparation of 320 samples in six days, opening the door to extensively understanding the effects of xenobiotic and biotic factors on our internal ecology.
| Item Type: | Article |
|---|---|
| Additional Information: | 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 Genomics Institute [OGI-156 and OGI-149], the Natural Sciences and Engineering Research Council of Canada [NSERC, grant No. 210034], and the Ontario Ministry of Economic Development and Innovation [ORF-DIG-14405 and project 13440]. |
| Uncontrolled Keywords: | gut microbiome,biobanking,functional responses,high-throughput in vitro assay,metaproteomics |
| Faculty \ School: | Faculty of Medicine and Health Sciences > Norwich Medical School |
| UEA Research Groups: | Faculty of Medicine and Health Sciences > Research Centres > Metabolic Health |
| Related URLs: | |
| Depositing User: | LivePure Connector |
| Date Deposited: | 02 Sep 2025 09:30 |
| Last Modified: | 02 Sep 2025 09:30 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/100253 |
| DOI: | 10.20517/mrr.2023.57 |
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