A streamlined high-throughput metaproteomic workflow for functional analysis of the rumen microbiome

ABSTRACT: This project describes the development, optimization, and evaluation of a streamlined and scalable metaproteomic workflow for the functional characterization of the rumen microbiome. Rumen samples represent a highly complex biological matrix, and existing metaproteomic protocols are often labor-intensive and difficult to scale, limiting their application in large experimental studies. In this study, key steps of the metaproteomic pipeline were systematically assessed, including microbial cell extraction, cell lysis, protein digestion strategy, and LC–MS/MS acquisition parameters. The optimized workflow integrates a minimized cell extraction protocol starting from 0.5 g of rumen material, mechanical cell lysis using bead-based disruption, in-solution tryptic digestion, and short-gradient nanoLC–MS/MS analysis on an Orbitrap Fusion Lumos Tribrid mass spectrometer operated with FAIMS. The workflow was applied to a large set of samples originating from in vitro rumen fermentations, enabling assessment of both biological and technical variability. Results demonstrate robust taxonomic and functional profiling of the rumen microbiome, with biological variation dominating over technical variation and median coefficients of variation in the range of 21–24%. Overall, this dataset provides a resource supporting high-throughput functional investigations of rumen microbial ecosystems.

INSTRUMENT(S):

ORGANISM(S): Bos Taurus (bovine)

TISSUE(S): Saliva

SUBMITTER: Ane Laura Fineid Pedersen  

LAB HEAD: Jana Seifert

PROVIDER: PXD078749 | Pride | 2026-08-05

REPOSITORIES: Pride