Benchmarking PASEF Mass Spectrometry Strategies for High-Sensitivity Gut Metaproteomics

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Data Quality: STRONG
Summary of  Nature Communications https://doi.org/10.1038/s41467-026-75845-5 
Dr. Feng Xian  et al.

Points

University of Vienna researchers systematically benchmarked five mass spectrometry PASEF acquisition modes across 540 LC-MS runs using human fecal samples.
Data-independent strategies including DIA-PASEF and Slice-PASEF outperformed conventional data-dependent acquisition modes in peptide coverage and low-abundance protein detection.
Advanced ion mobility acquisition demonstrated superior quantitative reproducibility, reduced ratio compression, and expanded functional annotation depth for complex gut microbial communities.
Biological validation in a murine colonic injury model showed concordant host repair and microbial functional changes during intestinal inflammation onset and recovery.
Metaproteomics enables direct measurement of active microbial and host protein expression, overcoming the functional limitations of traditional DNA-based genomic sequencing.

Summary

The functional expression of gut microbial communities by systematically benchmarking mass spectrometry acquisition modes to overcome sample complexity in metaproteomics. While genomic sequencing identifies potential microbial functions, metaproteomics reveals active microbial protein expression and host physiological responses. Led by researchers at the University of Vienna, the study benchmarked trapped ion mobility spectrometry combined with parallel accumulation–serial fragmentation (PASEF) to establish high-sensitivity, reproducible strategies for clinical and translational microbiome research.

To systematically evaluate acquisition performance, investigators benchmarked five distinct PASEF modes—data-dependent acquisition (DDA-PASEF), data-independent acquisition (DIA-PASEF), Slice-PASEF, Synchro-PASEF, and midia-PASEF. Utilizing a complex human fecal peptide background spiked with defined bacterial reference strains, the researchers analyzed 540 liquid chromatography–mass spectrometry (LC–MS) acquisitions across three chromatographic gradients and multiple input concentrations under matched experimental conditions.

Data-derived performance scores demonstrated that DIA-based strategies significantly outperformed conventional DDA-PASEF in peptide and protein coverage, particularly for low-abundance microbial features. Both DIA-PASEF and Slice-PASEF exhibited superior quantitative reproducibility, reduced ratio compression, expanded functional annotation depth, and consistent species-abundance scaling. In a murine colonic injury model, DIA- and Slice-PASEF captured highly concordant host tissue repair and microbial functional responses during intestinal inflammation onset and recovery, demonstrating that optimized metaproteomic acquisition translates to robust biological insights in gastrointestinal disease.

Link to the article: https://www.nature.com/articles/s41467-026-75845-5

References

Xian, F., Mitulovic, G., Ravi Kumar, R. K., Uhrik, L., Urbauer, E., Aguanno, D., Haller, D., Schmidt, M., & Gomez-Varela, D. (2026). Systematic evaluation of PASEF acquisition strategies in complex metaproteomes. Nature Communications, 17(1), 7406. https://doi.org/10.1038/s41467-026-75845-5