Expanding the Characterization of Microbial Ecosystems using DIA-PASEF Metaproteomics

Abstract

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 of mouse gut microbiota and, importantly, also of the host proteome. 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.

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

“…To obtain a comprehensive and confident proteotyping of complex metaproteomic samples, we set a multi-faceted strategy combining uMetaP powered by the extra mobility dimension provided by DDA-Parallel Acquisition Serial Fragmentation 26 (DDA-PASEF), the use of pH-fractionated mouse fecal microbiome peptide samples, and a database creation pipeline including validation and rescoring at an FDR 1% level. As shown in Figure 1A, uMetaP fragmented up to 4 times more precursors across all 8 fractions compared to a similar approach using the previous generation timsTOF Pro mass spectrometer, even if the latter was used with a longer gradient and 5 times more injected peptides.…”

MetaPep: A core peptide database for faster human gut metaproteomics database searches

“…Additionally, an in-house DIA-PASEF raw file was also searched against both the MetaPep peptide sequence database and the spectral library with DIA-NN [29] using default parameters to evaluate the performance of MetaPep on DIA-PASEF raw files. The methods for the acquisition of this in-house DIA-PASEF raw file were as described by Gomez-Varela et al [31] , with minor modifications as detailed in Supplementary Methods .…”