Systematic evaluation of PASEF acquisition strategies in complex metaproteomes

Metaproteomics measures functional expression in complex microbial communities, but extreme sample complexity and dynamic range challenge acquisition strategies. Trapped ion mobility spectrometry with parallel accumulation-serial fragmentation (PASEF) has expanded into multiple acquisition modes, yet systematic evaluations in high-complexity metaproteomes remain limited. Here, we benchmark five PASEF modes-DDA-, DIA-, Slice-, Synchro-, and midia-PASEF-using a complex fecal peptide background spiked with defined bacterial references. Across three gradients and input levels, 540 LC-MS acquisitions are analyzed under matched conditions. Based on data-derived performance scores, DIA-based strategies outperform DDA-PASEF in peptide and protein coverage, particularly for low-abundance microbial features. DIA- and Slice-PASEF show strong quantitative reproducibility, reduced ratio compression, and consistent species-abundance scaling, while functional profiling reveals expanded annotation depth. When tested in a murine colonic injury model, the two highest-scoring methods, DIA- and Slice-PASEF, capture concordant host and microbial responses.