Systematic evaluation of PASEF acquisition strategies in complex metaproteomes

Metaproteomics enables direct measurement of functional activity in complex microbial communities but remains technically challenging due to the extreme complexity and dynamic range of microbiome samples. Trapped ion mobility spectrometry coupled with parallel accumulation–serial fragmentation (PASEF) offers unique advantages in general proteomics and in the analysis of complex metaproteomes. In recent years, PASEF has diversified into multiple acquisition modes. However, the lack of systematic side-by-side evaluations under matched experimental conditions, together with the predominant use of low- to medium-complexity benchmark samples, hampers informed selection of acquisition strategies. Here, we benchmark five PASEF acquisition modes (DDA-, DIA-, Slice-, Synchro-, and midia-PASEF) using a complex fecal peptide background spiked with defined bacterial references. Performance was evaluated across three chromatographic gradients and input levels, comprising 540 LC–MS acquisitions processed using a single computational workflow. DIA-based strategies consistently achieved greater peptide and protein coverage than DDA-PASEF, particularly for low-abundance microbial features. Slice- and DIA-PASEF exhibited the lowest quantitative variability, minimal ratio compression, and the most consistent scaling of species abundances, while functional profiling revealed an expanded dynamic range of enzyme annotations. Application to a murine colonic injury model demonstrated that DIA- and Slice-PASEF capture highly concordant host and microbial responses. Together, this study provides a unified evaluation of PASEF acquisition strategies and illustrates how acquisition choices influence sensitivity, reproducibility, and functional resolution in proteomic analyses.