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Wang et al. Microbiome Res Rep 2024;3:39 https://dx.doi.org/10.20517/mrr.2024.21 Page 9 of 17

Figure 2. Comparison of identification and quantification abilities of DDA and DIA-PASEF approaches. Number of peptides identified by
DDA and DIA-PASEF (A) mass spectrometry methods. Number of protein groups identified by DDA and DIA-PASEF (B) mass
spectrometry methods. Number of quantitated protein groups in any, half, or all (Q0, Q50, and Q100, respectively) samples in each
group identified by DDA- or DIA-PASEF (C) mass spectrometry. For each figure panel, facet_grid function in ggplot2 was used to
generate different sections; DC groups are at the top and NC at the bottom, while DDA groups are on the left side and DIA on the right
side. DDA: Data-dependent acquisition; DIA: data-independent acquisition; PASEF: parallel accumulation-serial fragmentation; DC:
differential centrifugation; NC: non-differential centrifugation.

DIA-PASEF mass spectrometry methods for mouse metaproteomic samples from various preparations
provide more identifications with fewer missing values, enhancing the potential for quantitative analysis.

DIA-PASEF for non-enriched samples yielded the highest recovery of small proteins and
antimicrobial peptides
When conducting the database search, we included both microbiome and host protein sequences. When
looking at host proteins, DIA-PASEF mass spectrometry methods identified a higher number of host
proteins per sample than DDA; this outcome was anticipated, as DIA-PASEF identified more proteins.
Despite an increased diversity of host proteins identified in the DIA-PASEF dataset for each sample, the
relative abundance of host proteins per sample did not show an obvious difference between both data
acquisition and protein digestion methods [Supplementary Figure 3]. Differential centrifugation does not
appear to have a major impact on the identification count of host proteins or their relative abundances,
[30]
which is different from a previous study with human feces . This might be due to the relatively low
amount of host protein (less than 18% in abundance) in the feces of healthy mice that were used in this