Project description:Transcriptome profiles of an aerobic photosynthetic bacterium Roseobacter denitrificans OCh114 grown under different oxygen tension and light irradiation conditions were determined by NimbleGen Prokaryotic Expression array (12x135K).
Project description:An aerobic photosynthetic bacterium Roseobacter denitrificans OCh114 has two DNR- and one FNR-type transcriptional regulators, which are predicted to sense nitric oxide and oxygen, respectively. To investigate the role of these regulators in regulation of the denitrification genes, transcriptome profiles of mutant strains of R. denitrificans OCh114 deficient in the genes for the DNR- or FNR-type regulators were determined by NimbleGen Prokaryotic Expression array (12x135K).
Project description:Given the facilities for whole genome sequencing with next-generation sequencers, structural and functional gene annotation is now only based on automated prediction. However, errors in terms of gene structure are still frequently reported especially for the correct determination of initiation start codons. Here, we propose a strategy to enrich and detect protein N-termini by mass spectrometry in order to refine genome annotation. After selective protein N-termini derivatization using (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPPAc-OSu) as labeling reagent, protein digestion was performed with three proteases in parallel. TMPP-labeled N-terminal-most peptides were further resolved from internal peptides by the COmbined FRActional DIagonal Chromatography (COFRADIC) sorting methodology before analysis with tandem mass spectrometry. We refined the annotation of the genome of a model marine bacterium, Roseobacter denitrificans.
Project description:Proteogenomics is the alliance of proteomics and genomics with the aim of better annotating structural genes based on protein experimental pieces of evidence established by tandem mass spectrometry. While on average more than one tenth of N-termini of proteins are incorrectly annotated, there is a crucial need for methodological approaches to systematically establish translational starts of polypeptides and their maturations such as N-terminal methionine processing and peptide signal excision. Here, we describe a straightforward strategy to specifically label protein N-termini with a positively charged TMPP label, selectively capture these entities with in-house developed anti-TMPP antibodies coupled to magnetic beads, and analyze them by nanoLC-MS/MS. While most N-terminomics-oriented approaches are based on depletion of internal peptides to retrieve N-terminal peptides, this enrichment approach is fast and results highly specific for improved ionizable TMPP-labeled peptides. The whole proteome of the model marine bacterium Roseobacter denitrificans was analyzed.
