Dataset Information

A Method for Isolation and Proteomic Analysis of Outer Membrane Vesicles from Fecal Samples by LC-MS/MS.

ABSTRACT: Outer membrane vesicles (OMVs) are nanosized spheres secreted by bacteria that are similar to the vesicles known as exosomes, which are secreted by most mammalian cell types. In contrast to many studies focusing on optimizing methods for enriching exosomes from biological fluid, few studies have been conducted to investigate outer membrane vesicles from fecal samples. Herein, we have developed a pipeline comprised of membrane filtration and multiple cycles of ultracentrifugation (UC) to isolate OMVs from fecal samples for proteomics analysis, where multiple cycles of UC are required for removal of contaminants. By iTRAQ labeling quantitative proteomics analysis, different filter sizes (0.22 ?m and 0.45 ?m) were compared in terms of their performance in enriching OMVs and eliminating background fecal material. Using the 0.45 ?m filter, a slightly higher protein yield was obtained but no additional contaminating proteins from bacteria were identified compared to those from the 0.22 ?m filter. The 0.45 ?m filter together with the multiple cycles of UC were thus used to isolate OMVs for proteomics analysis. To our knowledge, this is the first study profiling a large number of OMV proteins from fecal samples. Such capabilities may help provide valuable information in understanding the communication between the host and microbiota, which is critical in preventing cancer and disease development.

SUBMITTER: Wu J

PROVIDER: S-EPMC6497448 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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A Method for Isolation and Proteomic Analysis of Outer Membrane Vesicles from Fecal Samples by LC-MS/MS.

Wu Jing J An Mingrui M Zhu Jianhui J Tan Zhijing Z Chen Grace Y GY Stidham Ryan W RW Lubman David M DM

Journal of proteomics & bioinformatics 20190318 2

Outer membrane vesicles (OMVs) are nanosized spheres secreted by bacteria that are similar to the vesicles known as exosomes, which are secreted by most mammalian cell types. In contrast to many studies focusing on optimizing methods for enriching exosomes from biological fluid, few studies have been conducted to investigate outer membrane vesicles from fecal samples. Herein, we have developed a pipeline comprised of membrane filtration and multiple cycles of ultracentrifugation (UC) to isolate ...[more]

PMID: 31057293

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Project description:Gram-negative bacteria constitutively release outer membrane vesicles (OMVs) during cell growth that play significant roles in bacterial survival, virulence and pathogenesis. In this study, comprehensive proteomic analysis of OMVs from a human gastrointestinal pathogen Campylobacter jejuni NCTC11168 was performed using high-resolution mass spectrometry. The OMVs of C. jejuni NCTC11168 were isolated from culture supernatants then characterized using electron microscopy and dynamic light scattering revealing spherical OMVs of an average diameter of 50nm. We then identified 134 vesicular proteins using high-resolution LTQ-Orbitrap mass spectrometry. Subsequent functional analysis of the genes revealed the relationships of the vesicular proteins. Furthermore, known N-glycoproteins were identified from the list of the vesicular proteome, implying the potential role of the OMVs as a delivery means for biologically relevant bacterial glycoproteins. These results enabled us to elucidate the overall proteome profile of pathogenic bacterium C. jejuni and to speculate on the function of OMVs in bacterial infections and communication.Biological significanceThis work demonstrates the importance of understanding vesicular proteomes from a human pathogen Campylobacter jejuni. From the secreted outer membrane vesicles (OMVs) of C. jejuni NCTC11168, we found a variety of virulence factors and essential proteins for bacterial survival. Bioinformatics analysis of these proteins predicted functional enrichment and localization. The most highly enriched were redox enzymes, which are considered to be essential for survival in oxygen-limiting environments and are predicted to be on the twin-arginine translocation (Tat) pathway suggesting a role for this pathway in the biogenesis of OMVs. This study additionally implicates a biological role for N-linked glycoproteins in OMVs. These approaches allow for a better understanding of the physiology of this important human pathogen.

Dataset Information

A Method for Isolation and Proteomic Analysis of Outer Membrane Vesicles from Fecal Samples by LC-MS/MS.

Publications

A Method for Isolation and Proteomic Analysis of Outer Membrane Vesicles from Fecal Samples by LC-MS/MS.

Similar Datasets

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