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Wang et al. Microbiome Res Rep 2024;3:39 https://dx.doi.org/10.20517/mrr.2024.21 Page 15 of 17

protein sequence database, including those indistinguishable proteins, improved identifications of both gut
microbial peptides and taxa. However, it is expected that the use of DDA-independent bioinformatics tools,
[43]
such as MetaDIA , and more comprehensive metagenomic assembled genome (MAG) databases, would
benefit the performance of metaproteomics and, thereby, the evaluations of the workflow.

Nevertheless, this study offered a comprehensive comparison of different experimental parameters,
including fecal sample pre-processing methods (differential centrifugation and non-differential
centrifugation), protein digestion techniques (in-solution and FASP with different molecular cut-off sizes),
data acquisition modes (DDA- and DIA-PASEF), and different bioinformatic workflows. We have
previously reported that lysis buffer and protein extraction protocols had major impacts on metaproteomics
observations, and the protein extraction protocols with strong detergent SDS and ultrasonication achieved
[32]
the best protein yields and peptide/protein identifications . Together with this previous study, we
highlighted the critical impact of experimental choices on metaproteomic outcomes and shed light on the
potential biases introduced at every step of the workflow. The outcomes of this study provide valuable
information in standardizing the metaproteomics workflow for applications, such as clinical study, drug
development, and regulatory assessment, especially for microbiome-based medicinal products.

DECLARATIONS
Acknowledgments
We gratefully acknowledge Drs. Simon Sauvé, Huixin Lu and Michael Rosu-Myles from Health Canada for
their critical comments on the manuscript.

Authors’ contributions
Made substantial contributions to the conception and design of the study and performed data analysis and
interpretation: Wang A, Fekete EEF, Zhang X
Performed data acquisition and contributed to data analysis and interpretation: Wang A, Fekete EEF, Zhang
X, Creskey M, Cheng K, Ning Z, Figeys D
Provided administrative, technical, and material support: Pfeifle A, Li X, Zhang X

Availability of data and materials
All MS proteomics data that support the findings of this study have been deposited to the ProteomeXchange
Consortium (http://www.proteomexchange.org) with the data set identifier PXD049086 and PXD049089.

Financial support and sponsorship
This work was supported by the Government of Canada through Health Canada.

Conflicts of interest
Figeys D co-founded MedBiome, a clinical microbiomics company. Zhang X is an Editorial Board member
of the journal Microbiome Research Reports, while the other authors have declared that they have no
conflicts of interest.

Ethical approval and consent to participate
The animal procedures were approved by the Animal Care Committee in Health Canada and performed in
accordance with institutional guidelines (Ethics Approval Number: 2020-011).

Consent for publication
Not applicable.