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Wang et al. Microbiome Res Rep 2024;3:39 https://dx.doi.org/10.20517/mrr.2024.21 Page 13 of 17

Figure 4. Taxonomic profiles of mouse fecal metaproteome. (A and B) Phylum level, (C and D) genus level with host (Mus), or (E and F)
without host were shown. The group mean relative abundance of phyla or genera was used for plotting. Top 5 phyla or top 10 genera
were shown, with the remainder grouped as “Other” in the stacked plots. Facet_grid function in ggplot2 was used to generate different
sections; DC groups are on the left side and NC on the right side. DC: Differential centrifugation; NC: non-differential centrifugation.

Differential centrifugation altered the functional profiles of fecal metaproteomes
Lastly, we evaluated the influence of sample preparation methods and MS acquisition on the functional
profiles obtained using fecal metaproteomics. We identified 24 out of the 26 Clusters of Orthologous Gene
(COG) categories for both DDA and DIA datasets with 1,356 and 1,261 COGs, respectively, in this study. As
shown in Figure 5, a consistent COG category-level composition of fecal metaproteome was observed
regardless of protein digestion method and MS acquisition mode. However, both DDA-PASEF and DIA-
PASEF datasets demonstrated that the differential centrifugation dramatically altered the observed
functional profiles in metaproteomics. These include the marked decrease in functional category J
(translation, ribosomal structure and biogenesis) and N (cell motility) by differential centrifugation, while
increased category E (amino acid transport and metabolism), M (cell wall/membrane/envelope biogenesis),
R (general function prediction only), and P (inorganic ion transport and metabolism). The decrease in
functions related to translation and cell motility suggests that the differential centrifugation may result in an
underestimation of microbial species with more active cell proliferation and higher motility. The functional