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.

Dual input control: activation of the Bartonella henselae VirB/D4 type IV secretion system by the stringent sigma factor RpoH1 and the BatR/BatS two‐component system

“…The dashed line pointing from BatR towards RpoH 1 refers to data from Omasits et al . (). The dashed lines pointing to the Trw T 4 SS reflect the fact that these effects are likely mediated by the KorA / KorB proteins, not represented in this model.…”

Proteogenomics: concepts, applications and computational strategies

“…Publicly available proteomic data may be mined to obtain protein-level evidence of expression of the novel transcripts nominated by genomics and transcriptomics technologies 10 . Furthermore, as generation of both transcriptomic and proteomic data in parallel is becoming increasingly common, there is an emerging trend of identifying peptides and proteins using proteomic data by matching MS/MS spectra against sample-specific protein sequence databases generated with the help of RNA-Seq (and/or ribosome profiling data) from the same samples 11–16 .…”

A Link between Arabinose Utilization and Oxalotrophy in Bradyrhizobium japonicum

“…After reduction and carbamidomethylation the proteins were digested with trypsin (Promega, Madison, WI, USA), and the resulting peptides were separated by reverse-phase high-performance liquid chromatography (RP-HPLC) and analyzed by a hybrid LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) interfaced with a nanoelectrospray source (12). Mass spectra were further processed with an in-house processing pipeline (26). In brief, fragment ion mass spectra were extracted from Thermo RAW files using msconvert (ProteoWizard, version 3.0.3831), merged in one MGF file per gel and searched against a composite B. japonicum USDA 110 protein database (RefSeq NC_004463.1, 22 July 2013) containing 256 common contaminants (e.g., human keratin and trypsin).…”