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

Up to date, most metaproteomic studies of the gut microbiota describe the use of 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 (NC). 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 non-microbial components (host and food) when compared to NC. Nevertheless, dramatic differences in the relative abundance of many gut microbial taxa were also observed, including a significant change in the Firmicutes/Bacteroidetes ratio. Furthermore, several microbial functional categories, including cell surface enzymes, membrane-associate proteins, and flagella, were significant 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.