RESUMO
We report the first proteomic analysis of the insoluble sub-proteome of the alkaliphilic and halotolerant deep-sea bacterium Oceanobacillus iheyensis HTE831. A multidimensional gel-based and gel-free analysis was utilised and a total of 4352 peptides were initially identified by automated MS/MS identification software. Automated curation of this list using PROVALT reduced our peptide list to 467 uniquely identified peptides that resulted in the positive identification of 153 proteins. These identified proteins were functionally classified and physiochemically characterised. Of 26 proteins identified as hypothetical conserved, we have assigned function to all but four. A total of 41 proteins were predicted to possess signal peptides. In silico investigation of these proteins allowed us to identify three of the five bacterial classes of signal peptide, namely: (i) twin-arginine translocation; (ii) Sec-type and (iii) lipoprotein transport. Our proteomic strategy has also allowed us to identify, at neutral pH, a number of proteins described previously as belonging to two putative transport systems believed to be of importance in the alkaliphilic adaptation of O. iheyensis HTE831.
Assuntos
Bacillaceae/metabolismo , Proteínas de Bactérias/metabolismo , Proteoma/metabolismo , Sequência de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
We report the first large-scale gel-free proteomic analysis of the soluble subproteome of the emerging pathogen Ochrobactrum anthropi. Utilizing our robust offline multidimensional protein identification protocol, a total of 57 280 peptides were initially identified utilizing automated MS/MS analysis software. We describe our investigation of the heuristic protein validation tool PROVALT and demonstrate its ability to increase the speed and accuracy of the curation process of large-scale proteomic datasets. PROVALT reduced our peptide list to 8517 identified peptides and further manual curation of these peptides led to a final list of 984 uniquely identified peptides that resulted in the positive identification of 249 proteins. These identified proteins were functionally classified and physiochemically characterized. A variety of typical "housekeeping" functions identified within the proteome included nucleic acid, amino and fatty acid anabolism and catabolism, glycolysis, TCA cycle, and pyruvate and selenoamino acid metabolism. In addition, a number of potential virulence factors of relevance to both plant and human disease were identified.
Assuntos
Proteínas de Bactérias/química , Ochrobactrum anthropi/química , Proteoma , Sequência de Aminoácidos , Automação , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Eletroforese em Gel Bidimensional/métodos , Espectrometria de Massas , Dados de Sequência Molecular , Ochrobactrum anthropi/genética , Ochrobactrum anthropi/patogenicidade , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Proteômica/métodosRESUMO
To further our understanding of the biology of the thermophilic bacterium Geobacillus thermoleovorans T80, we now report the first proteomic analysis of the insoluble subproteome of this isolate. A combination of both shotgun and multidimensional methodologies were utilized, and a total of 8628 peptides was initially identified by automated MS/MS identification software. Curation of these peptides led to a final list of 184 positive protein identifications. The proteins from this insoluble subproteome were functionally classified, and physiochemical characterization was carried out. Of 15 hypothetical conserved proteins identified, we have assigned function to all but four. A total of 31 proteins were predicted to possess signal peptides. In silico investigation of these proteins allowed us to identify four of the five bacterial classes of signal peptide, namely, (i) twin-arginine translocation; (ii) Sec-type; (iii) lipoprotein, and (iv) ABC transport. In addition, a number of proteins were identified that are known to be involved in the transport of compatible solutes, known to be important in microbial stress responses.
Assuntos
Bacillaceae/química , Proteínas de Bactérias/análise , Proteômica/métodos , Sequência de Aminoácidos , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Cromatografia Líquida , Biologia Computacional/métodos , Espectrometria de Massas , Dados de Sequência MolecularRESUMO
We report the first analysis of the soluble sub-proteome of the obligate thermophile, Geobacillus thermoleovorans T80, utilizing a robust multidimensional protein identification protocol. A total of 1,336 proteins were initially identified utilizing automated MS/MS identification software. Intensive manual curation resulted in a final list containing a total of 294 unique proteins. Physiochemical characterization and functional classification of the soluble sub-proteome was carried out. The strategy has allowed us to gain an insight into the cellular processes of this obligate thermophile, identifying a variety of proteins known to play a role in stress response. Included within these were a number of sigma factors such as sigma(A) that initiate transcription of the heat shock operons controlled by the HrcA-CIRCE complex within gram positive bacteria. In addition, it has enabled us to assign a degree of functionality to 29 out of 36 gene products detected in this study that were hitherto described as being only hypothetical conserved proteins.