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Page 14 of 17 Wang et al. Microbiome Res Rep 2024;3:39 https://dx.doi.org/10.20517/mrr.2024.21

Figure 5. Composition of identified COG categories in mouse fecal metaproteome. The group mean relative abundance of COG category
was plotted for (A) DDA and (B) DIA dataset, respectively. Each letter represents a COG category according to the Database of COGs
https://www.ncbi.nlm.nih.gov/research/cog/. COG: Clusters of Orthologous Gene; DDA: data-dependent acquisition; DIA: data-
independent acquisition.

profiles analysis again demonstrated that sample preparation methods need to be optimized for studies with
specific objectives or functional pathways of interest.

DISCUSSION
In this study, our findings showed that DIA acquisition provided a clear advantage compared to DDA for
identification and quantification of proteins, including small and antimicrobial proteins/peptides, in
microbiome samples. We also demonstrated that non-differential centrifugation methods improved the
recovery of small proteins and AMPs, and that FASP workflow using 10kDa molecular cut-off filter
achieved similar data outputs compared to in-solution digestion, both of which are commonly used in
proteomic and metaproteomic studies. While trying to provide a comprehensive comparison of different
experimental steps in metaproteomics, there are still limitations to be considered when continuing this
work. Firstly, this study used healthy mouse feces, which might not be representative of human feces, in
particular for diseased human fecal samples. To enable the assessment of multiple parameters, we used a
pooled mouse fecal sample and technical replicates in this study; the use of biological replicates for further
validation is needed and will provide more statistical power. Other sample types can also be tested, such as
intestinal content and aspirate samples. Secondly, the current bioinformatic workflow relies on the gene
catalog database and DDA data-generated spectral library or reduced protein database, which limits the
advantage of DIA-based metaproteomics. This study demonstrated that a library-free search with a full