Enrichment or depletion? The impact of stool pretreatment on metaproteomic characterization of the human gut microbiota

To date, most metaproteomic studies of the gut microbiota employ stool sample pretreatment methods to enrich for microbial components. However, a specific investigation aimed at assessing if, how, and to what extent this may impact on the final taxonomic and functional results is still lacking. Here, stool replicates were either pretreated by differential centrifugation (DC) or not centrifuged. Protein extracts were then processed by filter-aided sample preparation, single-run LC, and high-resolution MS, and the metaproteomic data were compared by spectral counting. DC led to a higher number of identifications, a significantly richer microbial diversity, as well as to reduced information on the nonmicrobial components (host and food) when compared to not centrifuged. Nevertheless, dramatic differences in the relative abundance of several gut microbial taxa were also observed, including a significant change in the Firmicutes/Bacteroidetes ratio. Furthermore, some important microbial functional categories, including cell surface enzymes, membrane-associated proteins, extracellular proteins, and flagella, were significantly reduced after DC. In conclusion, this work underlines that a critical evaluation is needed when selecting the appropriate stool sample processing protocol in the context of a metaproteomic study, depending on the specific target to which the research is aimed. All MS data have been deposited in the ProteomeXchange with identifier PXD001573 (http://proteomecentral.proteomexchange.org/dataset/PXD001573).