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Systematic evaluation of PASEF acquisition strategies in complex metaproteomes

ABSTRACT: Trapped ion mobility spectrometry coupled with parallel accumulation–serial fragmentation (PASEF) has enabled multiple acquisition strategies for proteomics, yet their comparative performance in complex metaproteomes remains unclear. Here we benchmark five modes—DDA-, DIA-, Slice-, Synchro- and midia-PASEF (PentaPASEF)—using human fecal samples spiked with defined bacterial references across three chromatographic gradients and input levels, comprising 540 LC–MS acquisitions. DIA-based strategies consistently delivered greater peptide and protein coverage than DDA-PASEF, particularly for low-abundance microbial features, and maintained stable performance under high-throughput conditions. Slice- and DIA-PASEF provided the lowest quantitative variability, minimal ratio compression and the most consistent species-level abundance scaling, while functional analysis revealed expanded dynamic range for DIA-based methods. Biological validation in a murine colonic epithelial injury model showed that DIA- and Slice-PASEF capture concordant host and microbial responses. This study establishes a unified reference for selecting PASEF acquisition strategies for sensitive and reproducible metaproteomics.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Colon, Feces

SUBMITTER: Feng Xian

LAB HEAD: Manuela Schmidt

PROVIDER: PXD073688 | Pride | 2026-07-03

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	DistalColon_metaP.7z	Other
	ProximalColon_metaP.7z	Other
	Search_output_DIA.parquet	Other
	Search_output_Slice.parquet	Other
	checksum.txt	Txt

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Systematic evaluation of PASEF acquisition strategies in complex metaproteomes

Project description:Trapped ion mobility spectrometry coupled with parallel accumulation–serial fragmentation (PASEF) has enabled multiple acquisition strategies for proteomics, yet their comparative performance in complex metaproteomes remains unclear. Here we benchmark five modes—DDA-, DIA-, Slice-, Synchro- and midia-PASEF (PentaPASEF)—using human fecal samples spiked with defined bacterial references across three chromatographic gradients and input levels, comprising 540 LC–MS acquisitions. DIA-based strategies consistently delivered greater peptide and protein coverage than DDA-PASEF, particularly for low-abundance microbial features, and maintained stable performance under high-throughput conditions. Slice- and DIA-PASEF provided the lowest quantitative variability, minimal ratio compression and the most consistent species-level abundance scaling, while functional analysis revealed expanded dynamic range for DIA-based methods.

2026-07-03 | PXD073779 | Pride

Expanding the Characterization of Microbial Ecosystems using DIA-PASEF Metaproteomics

Project description:Metaproteomics is gaining momentum in microbiome research due to the multi-dimensional information it provides. However, current approaches have reached their detection limits. We present a highly-sensitive metaproteomic workflow using the extra information captured by Parallel Accumulation-Serial Fragmentation (PASEF) technology. The comparison of different acquisition modes and data analysis software packages showed that DIA-PASEF and DIA-NN doubled protein identifications in the mouse gut microbiota and, importantly, also in the host proteome compared to DDA-PASEF. DIA-PASEF significantly improved peptide detection reproducibility and quantification accuracy, which resulted in more than twofold identified taxa, reaching depths comparable to metagenomic studies. Consequently, DIA-PASEF exhibited improved coverage of functional networks revealing 131 additional pathways compared to DDA-PASEF. We applied our optimized workflow to a pre-clinical mouse model of chronic pain, in which we deciphered novel host-microbiome interactions. In summary, we present here a metaproteomic approach that paves the way for increasing the functional characterization of microbiome ecosystems and is applicable to diverse fields of biological research.

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Deep Proteome profiling reveals signatures of age and sex differences in paw skin and sciatic nerve of naïve mice

Project description:The age and sex of studied animals profoundly impacts experimental outcomes in animal-based biomedical research. However, most preclinical studies in mice use a wide-spanning age range from 4 to 14 weeks and do not assess study parameters in male and female mice in parallel. This raises concerns regarding reproducibility and neglect of potentially relevant age and sex differences. Furthermore, the molecular setup of tissues in dependence of age and sex is unknown in naïve mice precluding efficient translational research. Here, we first compared two different mass spectrometric acquisition methods – DDA- and DIA-PASEF – in order to maximize the depth of proteome quantitation. We then employed an optimized workflow of quantitative proteomics based on DIA-PASEF followed by DIA-NN data analysis, and revealed significant differences in mouse paw skin and sciatic nerve (SCN) when comparing (i) male and female mice, and, in parallel, (ii) adolescent mice (4 weeks) with adult mice (14 weeks).

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prm-PASEF and g-dia-PASEF analysis of plasma samples from colorectal cancer and control patients

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Comprehensive Proteomics Profiling of Zebrafish Retina Using a Hybrid DDA/DIA-PASEF Spectral Library on a timsTOF Pro 2 Mass Spectrometer

Project description:The zebrafish (Danio rerio) retina has been increasingly used for studying vertebrate visual processing and ocular growth regulation as it has a high anatomical and physiological similarity to the human eye. Recently, the integration of trapped ion mobility spectrometry (TIMS) with parallel accumulation–serial fragmentation (PASEF), has emerged as a powerful approach for proteomics analysis. In this study, a retina specific hybrid spectral library was constructed for quantitative proteomics analysis of the zebrafish retina. We integrated data dependent acquisition PASEF (DDA PASEF) and data independent acquisition PASEF (DIA PASEF) datasets acquired from 16 individual retinal protein lysates on a Bruker timsTOF Pro 2 mass spectrometer to generate the hybrid spectral library. The Pulsar search engine in Spectronaut 22 was used for hybrid library generation. The resulting hybrid library comprised 8,551 precursors, 7,866 unique peptides, and 1,494 protein groups (1% FDR), representing 44.5% of the reviewed zebrafish reference proteome.16 DIA PASEF runs from individual samples were then analyzed in both library based (using the hybrid library) and library free settings in Spectronaut (version 22) and DIA NN (version 2.3.2). It demonstrated that the hybrid library outperformed library free approaches, with improved peptide and precursor identifications across both proteomics platforms.

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2024-05-31 | PXD047793 | Pride

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