RT Journal
A1 Omasits, Ulrich
A1 Quebatte, Maxime
A1 Stekhoven, Daniel J.
A1 Fortes, Claudia
A1 Roschitzki, Bernd
A1 Robinson, Mark D.
A1 Dehio, Christoph
A1 Ahrens, Christian H.
T1 Directed shotgun proteomics guided by saturated RNA-seq identifies a complete expressed prokaryotic proteome
JF Genome Research 
JO Genome Research 
YR 2013 
FD November 01 
VO 23 
IS 11 
SP 1916 
OP 1927 
DO 10.1101/gr.151035.112 
UL http://genome.cshlp.org/content/23/11/1916.abstract 
AB 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.