A Proteomic Approach to Quantifying the Mass Concentration of Cellulase Enzymes Produced by Clostridium thermocellum

To accurately assess rates of microbial cellulose utilization (MCU), it is essential to independently determine cell, cellulase and cellulose mass concentration. Such ability would enable us to directly address questions related to substrate utilization, allocation of cellular resources between cell and cellulase synthesis, cell- and cellulase-specific cellulose hydrolysis rates and bioenergetics. Current methodologies for cellulase determination involve work-intensive purification procedures. In this study we seek to develop and validate a method for cellulase determination which involves minimal manipulation of a fermentation sample. Using proteomic protein determination, we seek to reliably and robustly predict mass concentration of cellulase across varying growth conditions, substrates and cellulase types (cell free vs. cell associated). Our goal was to identify a core group of cellulosomal proteins from Clostridium thermocellum which can be assayed using proteomics to determine total cellulosomal protein. Ten proteins that comprise approximately 90% of total cellulosomal proteins have been identified in Clostridium thermocellum fermentations. From these ten proteins, 40 peptides have been selected for targeted analysis to determine cellulase mass concentration in cell digest samples. Good candidate peptide sequences were selected to use for quantification based on Mudpits, LTQ and triple quadrupole MS measurements. We analyzed the candidate proteins for variability in the fraction of total cellulosomal mass represented in samples from varying conditions. We optimized peptide selection for these protein components ensuring representative unique peptides with good signal quality for all proteins of interest were selected. We also examined the relationship between proteomic determination of total cellulase based on the peptides selected for analysis and protein determination using conventional protein measurement techniques like the Bradford assay. Once we established the core group, good peptides that could reproducibly be used to quantify them, and the correlation between the protein determination methods, a prediction capability curve was established to determine the total amount of cellulase in a sample.