Dataset Information

Host-centric proteomics of stool

ABSTRACT: Soluble extra-cellular proteins and peptides from human and mouse stool were fractionated on a C-4 solid phase extraction cartidge, trypsin digested, C-18 clean up and identified with MS/MS on a LTQ-Velos Orbitrap. Data processing: The readRAW program was used to generate peak lists from the original data. All protein sequences were reversed and concatenated to the original forward-oriented proteins. Spectra were assigned to peptides using a semi-specific enzyme specificity with the Mascot search algorithm (version 2.3.01) using static carbamidomethylation of cysteines, differential oxidized methionines, 50ppm precursor mass tolerance, 2 miscleavages and 0.8Da fragment ion tolerance. Searched against a mouse or human database.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

SUBMITTER: Joshua Lichtman

PROVIDER: PXD000240 | Pride | 2013-10-16

REPOSITORIES: Pride

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Publications

Host-centric proteomics of stool: a novel strategy focused on intestinal responses to the gut microbiota.

Lichtman Joshua S JS Marcobal Angela A Sonnenburg Justin L JL Elias Joshua E JE

Molecular & cellular proteomics : MCP 20130827 11

The diverse community of microbes that inhabits the human bowel is vitally important to human health. Host-expressed proteins are essential for maintaining this mutualistic relationship and serve as reporters on the status of host-microbiota interaction. Therefore, unbiased and sensitive methods focused on host proteome characterization are needed. Herein we describe a novel method for applying shotgun proteomics to the analysis of feces, focusing on the secreted host proteome. We have conducted ...[more]

PMID: 23982161

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Project description:Understanding gene expression by bacteria during the actual course of human infection may provide important insights into microbial pathogenesis. In this study, we evaluated the transcriptional profile of Vibrio cholerae, the causative agent of cholera, in clinical specimens from cholera patients. We collected samples of human stool and vomitus that were positive by dark-field microscopy for abundant vibrios and used a microarray to compare gene expression in organisms recovered directly from the early and late stages of human infection. Our results reveal that V. cholerae gene expression within the human host environment differs from patterns defined in in vitro models of pathogenesis. tcpA, the major subunit of the essential V. cholerae colonization factor, was significantly more highly expressed in early compared with late infection; however, the genes encoding cholera toxin were not highly expressed in either phase of human infection. Furthermore, expression of the virulence regulators, toxRS and tcpPH, was uncoupled. Interestingly, the pattern of gene expression indicates that the human upper intestine may be a uniquely suitable environment for the transfer of genetic elements that are important in the evolution of pathogenic strains of V. cholerae. These findings provide a more detailed assessment of the transcriptome of V. cholerae in the human host than previous studies of organisms in stool alone and have implications for cholera control and the design of improved vaccines. The V. cholerae microarray consists of 3,890 full-length PCR products representing the annotated open reading frames from the initial release of the V. cholerae N16961 genome. Each labeling and hybridization was performed in duplicate. Genomic DNA was used as a universal internal control for the quality of the microarray and to allow for the comparison of results across multiple experiments. Data were normalized using locally-weighted regression (Lowess) to obtain the relative abundance of each transcript as an intensity ratio with respect to that of genomic DNA. High correlation coefficients were observed between technical replicates (Pearsonâs correlation coefficient (r) > 0.80) and between results of separate clinical specimens of vomitus (r > 0.77) and of stool (r > 0.80). Hence, the results from the two clinical vomitus specimens and the five clinical stool specimens were pooled. Fold changes for the relative expression of a given gene between the two clinical specimens were calculated by dividing the normalized median intensity ratios with respect to genomic DNA.

Dataset Information

Host-centric proteomics of stool

Publications

Host-centric proteomics of stool: a novel strategy focused on intestinal responses to the gut microbiota.

Similar Datasets

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