Tuesday, May 1, 2012
Napoleon Ballroom C-D, 3rd fl (Sheraton New Orleans)
Differential shotgun proteome analysis of a mesophilic, hydrogen and ethanol producing cellulolytic bacterium, Clostridium termitidis, was performed using two growth substrates. Batch experiments were carried out in replicate serum bottles (50 mL) containing 1191 media with cellobiose and alpha-cellulose substrates at 37 0C. Active cultures of C. termitidis were made by two consecutive passages on 2 g/L alpha-cellulose or cellobiose, correspondingly, with 10% v/v inoculation. Liquid samples were collected during the mid-log phase, and the pellets were separated by centrifugation at 4,700 rpm using Sorvall SH BK-3000 rotor. Total proteins were extracted using SDT-lysis method and after trypsin digestion, the resulting peptides were desalted and cleaned using Agilent Technologies RP HPLC C18 column. After desalting, 100 micrograms of total peptides from both the biological replicates were labeled using isobaric tags for Relative/Absolute Quantitation (iTRAQ), and subjected to 2D LC/MS/MS analysis using an AB SCIEX QSTAR® Elite Hybrid TOF-MS system. An in-house GPU-based search engine scanned the fragment ion spectra against a C. termitidis derived peptide database to report observed peptide sequences and their corresponding iTRAQ reporter-ion intensity values. Approximately, 1370 proteins were identified with replicate-correlated protein level Z-score values, yielding relative protein expression levels of alpha-cellulose versus cellobiose grown cultures. To aid further analysis, a tentative COG map was constructed based on identified C. termitidis protein sequence homologies against three related microorganims: C. cellulolyticum H10, C. phytofermentans ISDg, and C. thermocellum ATCC 27405.