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Page 2 of 17 Wang et al. Microbiome Res Rep 2024;3:39 https://dx.doi.org/10.20517/mrr.2024.21

Compared to DDA, DIA-PASEF identified more microbial peptides, quantified more proteins with fewer missing
values, and recovered more small antimicrobial proteins. We did not observe any obvious impacts of protein
digestion methods on both taxonomic and functional profiles. However, differential centrifugation decreased the
recovery of small and antimicrobial proteins, biased the taxonomic observation with a marked overestimation of
Muribaculum species, and altered the measured functional compositions of metaproteome.

Conclusion: This study underscores the critical impact of experimental choices on metaproteomic outcomes and
sheds light on the potential biases introduced at different stages of the workflow. The comprehensive
methodological comparisons serve as a valuable guide for researchers aiming to enhance the accuracy and
completeness of metaproteomic analyses.
Keywords: Fecal metaproteomics, microbiome, mass spectrometry, differential centrifugation

INTRODUCTION
The human gut microbiome contains an estimated 100 trillion microorganisms, including bacteria, fungi,
protozoa, and viruses, which interact with each other and their host to foster a complex and dynamic
environment . The symbiotic host-microbial relationship of the gut microbiome is crucial to human
[1-3]
[4]
health and contributes to many biological processes, such as metabolism, immunomodulation, etc. . Many
studies also suggest that microbiome dysbiosis is correlated with and may lead to the development of
neurodegenerative, cardiovascular, metabolic, and gastrointestinal diseases, among others [3,5-7] . With the
emerging importance of the gut microbiome in human health, disease, and therapeutics, studies on the
[8]
microbiome, its taxa, and its products have become increasingly significant .

Given the extremely high complexity of the microbiome, meta-omics approaches including metagenomics,
metatranscriptomics, metabolomics, and metaproteomics, are commonly used in studying the microbiome
composition and functions [9,10] . Among the different omics approaches, metaproteomics uses a mass
spectrometer to directly measure the protein expressions and post-translational modifications (PTMs) of
[11]
the microbial community . Mass spectrometry (MS) analysis can be conducted with a data-dependent
acquisition (DDA) or data-independent acquisition (DIA) strategy. DDA-based metaproteomics is
commonly used due to its easy setup and analysis, flexibility, breadth of detection, and ability to relatively
[12]
quantify chemically labeled peptides . In a DDA mode, the most abundant ions from MS1 scan will be
selected and fragmented during tandem MS scans; however, this data acquisition mode can risk losing
information on the other less abundant peptides, particularly in complex samples such as microbiomes,
which limits the depth, sensitivity, and reproducibility of metaproteomic data [13,14] . Contrastingly, DIA can
sample all the peptides within the selected mass range and, therefore, theoretically can detect lower-
abundance peptides in complex samples . In the past few years, the application of DIA-MS-based
[15]
proteomics was profoundly expanded due to these advantages, the advancement of bioinformatics tools,
such as DIA-NN , and advanced instrumental developments, such as timsTOF Pro and Astral MS
[16]
[17]
analyzer . More recently, the application of DIA-MS in metaproteomics has been reported, demonstrating
[18]
great potential in increasing the depth of identification and accuracy of quantifications [19-21] .
One advantage of metaproteomics is the capability to measure non-bacterial components, including
proteins originating from the host, as well as viral, fungal and archaeal species, without the need for
additional experimental efforts . This is particularly ideal for trans-kingdom, host-microbiome interaction
[22]
studies [23,24] . The intestinal lumen is the home of diverse biotic and abiotic components, including host-
secreted proteins such as antimicrobial proteins and proteins produced by microbes themselves, such as
small microbial proteins. These small proteins/polypeptides, including a high proportion of antimicrobial