Assessing fecal metaproteomics workflow and small protein recovery using DDA and DIA PASEF mass spectrometry

Aim: This study aims to evaluate the impact of experimental workflow on fecal metaproteomic observations, including the recovery of small and antimicrobial proteins often overlooked in metaproteomic studies. The overarching goal is to provide guidance for optimized metaproteomic experimental design, considering the emerging significance of the gut microbiome in human health, disease, and therapeutic interventions. Methods: Mouse feces were utilized as the experimental model. Fecal sample pre-processing methods (differential centrifugation and non-differential centrifugation), protein digestion techniques (in-solution and filter-aided), data acquisition modes (data-dependent and data-independent, or DDA and DIA) when combined with parallel accumulation-serial fragmentation (PASEF), and different bioinformatic workflows were assessed. Results: We showed that, in DIA-PASEF metaproteomics, the library-free search using protein sequence database generated from DDA-PASEF data achieved better identifications than using the generated spectral library.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.