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Directed shotgun proteomics guided by saturated RNA-seq identifies a complete expressed prokaryotic proteome


Omasits, Ulrich; Quebatte, Maxime; Stekhoven, Daniel J; Fortes, Claudia; Roschitzki, Bernd; Robinson, Mark D; Dehio, Christoph; Ahrens, Christian H (2013). Directed shotgun proteomics guided by saturated RNA-seq identifies a complete expressed prokaryotic proteome. Genome Research, 23(11):1916-1927.

Abstract

Prokaryotes are, due to their moderate complexity, particularly amenable to the comprehensive identification of the protein repertoire expressed under different conditions. We applied a generic strategy to identify a complete expressed prokaryotic proteome, which is based on the analysis of RNA and proteins extracted from matched samples. Saturated transcriptome profiling by RNA-seq provided an endpoint estimate of the protein-coding genes expressed under two conditions which mimic the interaction of Bartonella henselae with its mammalian host. Directed shotgun proteomics experiments were carried out on four subcellular fractions. By specifically targeting proteins which are short, basic, low abundant and membrane localized, we could eliminate their initial under-representation compared to the estimated endpoint. A total of 1,250 proteins were identified with an estimated false discovery rate below 1%. This represents 85% of all distinct annotated proteins and about 90% of the expressed protein-coding genes. Genes that were detected at the transcript but not protein level, were found to be highly enriched in several genomic islands. Furthermore, genes that lacked an ortholog and a functional annotation were not detected at the protein level; these may represent examples of over-prediction in genome annotations. A dramatic membrane proteome re-organization was observed including differential regulation of autotransporters, adhesins and hemin binding proteins. Particularly noteworthy was the complete membrane proteome coverage, which included expression of all members of the VirB/D4 type IV secretion system, a key virulence factor.

Abstract

Prokaryotes are, due to their moderate complexity, particularly amenable to the comprehensive identification of the protein repertoire expressed under different conditions. We applied a generic strategy to identify a complete expressed prokaryotic proteome, which is based on the analysis of RNA and proteins extracted from matched samples. Saturated transcriptome profiling by RNA-seq provided an endpoint estimate of the protein-coding genes expressed under two conditions which mimic the interaction of Bartonella henselae with its mammalian host. Directed shotgun proteomics experiments were carried out on four subcellular fractions. By specifically targeting proteins which are short, basic, low abundant and membrane localized, we could eliminate their initial under-representation compared to the estimated endpoint. A total of 1,250 proteins were identified with an estimated false discovery rate below 1%. This represents 85% of all distinct annotated proteins and about 90% of the expressed protein-coding genes. Genes that were detected at the transcript but not protein level, were found to be highly enriched in several genomic islands. Furthermore, genes that lacked an ortholog and a functional annotation were not detected at the protein level; these may represent examples of over-prediction in genome annotations. A dramatic membrane proteome re-organization was observed including differential regulation of autotransporters, adhesins and hemin binding proteins. Particularly noteworthy was the complete membrane proteome coverage, which included expression of all members of the VirB/D4 type IV secretion system, a key virulence factor.

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23 citations in Web of Science®
24 citations in Scopus®
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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Functional Genomics Center Zurich
07 Faculty of Science > Institute of Molecular Life Sciences
Special Collections > SystemsX.ch > Research, Technology and Development Projects > InfectX
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:2013
Deposited On:16 Sep 2013 06:56
Last Modified:05 Apr 2016 16:58
Publisher:Cold Spring Harbor Laboratory Press
ISSN:1088-9051
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1101/gr.151035.112
PubMed ID:23878158

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