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

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. DOI: https://doi.org/10.1101/gr.151035. 112 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-80861 Akzeptierte Version Originally published at: 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. DOI: https://doi.org/10.1101/gr.151035. 112 10.1101/gr.151035. 112 Access the most recent version at doi: published online July 22, 2013 Genome Res. Ulrich Omasits, Maxime Quebatte, Daniel J. Stekhoven, et al. identifies a complete expressed prokaryotic proteome Directed shotgun proteomics guided by saturated RNA-seq Material Supplemental http://genome.cshlp. org/content/suppl/2013/08/20/gr.151035. 112.DC1. html P<P Published online July 22, 2013 in advance of the print journal. Preprint Accepted likely to differ from the final, published version. Peer-reviewed and accepted for publication but not copyedited or typeset; preprint is License Commons Creative . http://creativecommons.org/licenses/by-nc/3.0/ described at a Creative Commons License (Attribution-NonCommercial 3.0 Unported), as ). After six months, it is available under http://genome.cshlp. org/site/misc/terms.xhtml first six months after the full-issue publication date (see This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the Service Email Alerting click here. top right corner of the article or Receive free email alerts when new articles cite this article sign up in the box at the object identifier (DOIs) and date of initial publication. by PubMed from initial publication. Citations to Advance online articles must include the digital publication). Advance online articles are citable and establish publication priority; they are indexed appeared in the paper journal (edited, typeset versions may be posted when available prior to final Advance online articles have been peer reviewed and accepted for publication but have not yet http://genome.cshlp. org/subscriptions go to: Genome Research To subscribe to © 2013, Published by Cold Spring Harbor Laboratory Press Cold Spring Harbor Laboratory Press on September 15, 2013 Published by genome.cshlp. org Downloaded from