Dataset Information

Rumen metaproteomics - Catching the tip of the iceberg - evaluation of sample preparation protocols for metaproteomic studies of the rumen microbiota

ABSTRACT: Metaproteomic studies of the rumen microbiota are challenged by the need of optimized sample preparation protocols in order to retrieve an enhanced amount of prokaryotic instead of plant and bovine derived cells before protein extraction and subsequent LC-MS/MS analysis. The present study evaluates three different protocols applied to the rumen microbiota either attached to plant fibres or present as planktonic cells. The findings of our work suggest the integration of cheesecloth-gauze filtration in sample preparation to achieve a better protein identification ratio.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Archaea Bacteria Eukaryota (eucaryotes)

SUBMITTER: Jana Seifert

LAB HEAD: Jana Seifert

PROVIDER: PXD001526 | Pride | 2015-04-01

REPOSITORIES: Pride

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Catching the tip of the iceberg - evaluation of sample preparation protocols for metaproteomic studies of the rumen microbiota.

Deusch Simon S Seifert Jana J

Proteomics 20150512 20

Various metabolic processes are performed in the rumen caused by a complex microbiota comprising bacteria, archaea, protozoa, and fungi. Thus, the description of the active microbial fraction and their functions are of great interest for animal nutrition, biotechnology, and climatology. Metaproteomic studies of the rumen microbiota are challenged by the need of optimized sample preparation protocols in order to retrieve an enhanced amount of prokaryotic instead of plant- and bovine-derived cells ...[more]

PMID: 25765363

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Project description:The structure and function of the microbiome inhabiting the rumen are, amongst other factors, mainly shaped by the animal’s feed intake. Describing the influence of different diets on the inherent community arrangement and associated metabolic activities of the most active ruminal fractions (bacteria and archaea) is of great interest for animal nutrition, biotechnology and climatology. Samples were obtained from three fistulated Jersey cows rotationally fed with corn silage, grass silage or hay, each supplemented with a concentrate mixture. Samples were fractionated into ruminal fluid, squeezed solid and solid matter. DNA, proteins and metabolites were analyzed subsequently. DNA extracts were used for Illumina sequencing of the 16S rRNA gene and the metabolomes of rumen fluids were determined by 500MHz-NMR spectroscopy. Tryptic peptides derived from protein extracts were measured by LC-ESI-MS/MS and spectra were processed by a two-step database search for quantitative metaproteome characterization. Protein- and DNA-based datasets revealed significant differences between sample fractions and diets and affirmed similar trends concerning shifts in phylogenetic composition. Ribosomal genes and proteins belonging to the phylum of Proteobacteria, particularly Succinivibrionaceae, exhibited a higher abundance in corn silage-based samples while fiber-degraders of the Lachnospiraceae family emerged in great quantities throughout the solid phase fractions. The analysis of 8163 quantified bacterial proteins revealed the presence of 166 carbohydrate active enzymes in varying abundance. Cellulosome affiliated proteins were less expressed in the grass silage, glycoside hydrolases appeared in slightest numbers in the corn silage. Most expressed glycoside hydrolases belonged to families 57 and 2. Enzymes analogous to ABC transporters for amino acids and monosaccharides were more abundant in the corn silage whereas oligosaccharide transporters showed a higher abundance in the fiber-rich diets. Proteins involved in carbon metabolism were detected in high numbers and identification of metabolites like short-chain fatty acids, methylamines and phenylpropionate by NMR enabled linkage between producers and products. This study forms a solid basis to retrieve deeper insight into the complex network of gut microbial adaptation.

			Action	DRS
	A1-5_RF_ARC.prot.xml	Xml
	A1-5_RF_BAC.prot.xml	Xml
	A1-5_RF_EUK.prot.xml	Xml
	A1-5_RSC_ARC.prot.xml	Xml
	A1-5_RSC_BAC.prot.xml	Xml