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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.

INSTRUMENT(S):

ORGANISM(S): Ligilactobacillus Murinus Dsm 20452 = Nbrc 14221 Homo Sapiens (human) Salinibacter Ruber Dsm 13855

SUBMITTER: Feng Xian

LAB HEAD: Manuela Schmidt

PROVIDER: PXD073779 | Pride | 2026-07-03

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	DDA.rar	Other
	DDA_22min_peptides.tsv	Tabular
	DDA_45min_peptides.tsv	Tabular
	DDA_5min_peptides.tsv	Tabular
	DIA.rar	Other

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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. 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.

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2023-05-04 | PXD040947 | Pride

Ultra-sensitive metaproteomics (uMetaP) redefines the “dark” metaproteome, enables single-bacterium resolution, and discovers hidden functions in the gut microbiome

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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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Project description:Data-independent acquisition (DIA) methods have become increasingly attractive in mass spectrometry (MS)-based proteomics, because they enable high data completeness and a wide dynamic range. Recently, we combined DIA with parallel accumulation – serial fragmentation (dia-PASEF) on a Bruker trapped ion mobility separated (TIMS) quadrupole time-of-flight (TOF) mass spectrometer. This requires alignment of the ion mobility separation with the downstream mass selective quadrupole, leading to a more complex scheme for dia-PASEF window placement compared to DIA. To achieve high data completeness and deep proteome coverage, here we employ variable isolation windows that are placed optimally depending on precursor density in the m/z and ion mobility plane. This Automatic Isolation Design procedure is implemented in the freely available py_diAID package. In combination with in-depth project-specific proteomics libraries and the Evosep LC system, we reproducibly identified over 7,700 proteins in a human cancer cell line in 44 minutes with quadruplicate single-shot injections at high sensitivity. Even at a throughput of 100 samples per day (11 minutes LC gradients), we consistently quantified more than 6,000 proteins in mammalian cell lysates by injecting four replicates. We found that optimal dia-PASEF window placement facilitates in-depth phosphoproteomics with very high sensitivity, quantifying more than 35,000 phosphosites in a human cancer cell line stimulated with an epidermal growth factor (EGF) in triplicate 21 minutes runs. This covers a substantial part of the regulated phosphoproteome with high sensitivity, opening up for extensive systems-biological studies.

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