title: Directed shotgun proteomics guided by saturated RNA-seq identifies a complete expressed prokaryotic proteome.
abstract: Prokaryotes, due to their moderate complexity, are 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 underrepresentation compared to the estimated endpoint. A total of 1250 proteins were identified with an estimated false discovery rate below 1%. This represents 85% of all distinct annotated proteins and ∼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 overprediction in genome annotations. A dramatic membrane proteome reorganization 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.
authors.affiliation: Quantitative Model Organism Proteomics, Institute of Molecular Life Sciences, University of Zurich, 8057 Zurich, Switzerland;
authors.forename: Ulrich
authors.initials: U
authors.lastname: Omasits
authors.forename: Maxime
authors.initials: M
authors.lastname: Quebatte
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Stekhoven
authors.forename: Claudia
authors.initials: C
authors.lastname: Fortes
authors.forename: Bernd
authors.initials: B
authors.lastname: Roschitzki
authors.forename: Mark D
authors.initials: MD
authors.lastname: Robinson
authors.forename: Christoph
authors.initials: C
authors.lastname: Dehio
authors.forename: Christian H
authors.initials: CH
authors.lastname: Ahrens
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D018416:Bartonella henselae / Q000235:genetics*; D001483:Base Sequence; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D016680:Genome, Bacterial; D008954:Models, Biological; D058977:Molecular Sequence Annotation; D020543:Proteome / Q000235:genetics* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D037521:Virulence Factors / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D020543:Proteome; D037521:Virulence Factors