Workflow for quantitative proteomic analysis of Clostridium acetobutylicum ATCC 824 using iTRAQ tags.
Methods
; 61(3): 269-76, 2013 Jun 15.
Article
em En
| MEDLINE
| ID: mdl-23523702
ABSTRACT
Clostridium acetobutylicum (Cac) is an anaerobic, endospore-forming, Gram-positive bacterium with tremendous promise for use as a biocatalyst for the production of fuels and solvents. Cac proteomic sample preparation for shotgun analysis typically involves a multitude of reagents for harsh lysis conditions and to maintain protein solubility. We describe a protein extraction and preparation method for Cac that is compatible with proteomic shotgun analysis using isobaric labeling approaches. The method is applied to the analysis of Cac grown under butanol stress and labeled using iTRAQ 4-plex reagents. This method relies on the use of calcium carbonate to facilitate lysis by sonication and a commercially available kit to remove detergents prior to labeling. This workflow resulted in the identification and quantitation of 566 unique proteins using ProteinPilot software with a false discovery rate of 0.01% for peptide matches and 0.70% for protein matches. Ninety-five proteins were found to have statistically higher expression levels in butanol-stressed Cac as compared to non-stressed Cac. Sixty-one proteins were found to have statistically lower expression levels in stressed versus non-stressed cells. This method may be applicable to other Gram-positive organisms.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Peptídeos
/
Proteínas de Bactérias
/
Proteoma
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Proteômica
/
Clostridium acetobutylicum
Idioma:
En
Revista:
Methods
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2013
Tipo de documento:
Article
País de afiliação:
Estados Unidos