1-26: Relative protein expression profiles and growth phase-dependent changes in protein expression in Clostridium thermocellum using 2D-HPLC-MS/MS

Tuesday, May 1, 2012
Napoleon Ballroom C-D, 3rd fl (Sheraton New Orleans)
Thomas Rydzak1, Peter McQueen2, Oleg V. Krokhin2, Vic Spicer3, Peyman Ezzati2, Ravi C. Dwivedi2, Dimitry Shamshurin2, Marina Rambler2, David Levin4, John A. Wilkins2 and Richard Sparling1, (1)Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada, (2)Department of Internal Medicine, University of Manitoba, Winnipeg, MB, Canada, (3)Department of Physics & Astronomy, University of Manitoba, Winnipeg, MB, Canada, (4)Biosystems Engineering, University of Manitoba, Winnipeg, MB, Canada
Clostridium thermocellum is an attractive candidate for biofuels production, producing H2, ethanol, and concomitant volatile fatty acids directly from cellulosic biomass.  2D-HPLC-MS/MS detected 1602 proteins of 3236 protein encoding genes with a 99.9% confidence score in cellobiose-grown exponential phase batch culture lysates.  Relative abundance profiles based on rI/Mr (# of redundant peptides hits divided by molecular mass of protein) revealed differences in protein levels of enzymes capable of catalyzing parallel pathways.  The majority of catabolic proteins suspected to be involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech hydrogenase, and NADH:ferredoxin oxidoreductase.  2D-HPLC-MS/MS analysis of iTRAQ labelled exponential and stationary phase lysates revealed growth-phase dependent changes in expression of 406 proteins.  Generally, expression of proteins involved in translation, transcription, and lipid metabolism decreased in stationary phase, whereas proteins involved in signal transduction, cell wall biogenesis, cell motility, energy production and conversion, and carbohydrate, amino acid, and nucleotide transport and metabolism increased in stationary phase by more that 1.4-fold.  Of the major proteins directly involved in conversion of cellobiose to end-products, expression of 1,3-phosphoglycerate kinase, enolase, two putative phosphoglyceraste mutases, pyruvate:ferredoxin oxidoreductase, acetate kinase, and an number of alcohol dehyrogenases increased in stationary phase, while a subunit of oxaloacetate decarboxylase and pyruvate phosphate dikinase decreased by more than 1.4-fold in stationary phase.  Multiple reaction monitoring-mass spectrometry (MRM) methods are being developed to detect major catabolic proteins for high throughput analysis under different growth conditions, including growth under high hydrogen.
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