A targeted proteomics toolkit for high-throughput absolute quantification of Escherichia coli proteins

Transformation of engineered Escherichia call into a robust microbial factory is contingent on precise control of metabolism. Yet, the throughput of omics technologies used to characterize cell components has lagged far behind our ability to engineer novel strains. To expand the utility of quantitative proteomics for metabolic engineering, we validated and optimized targeted proteomics methods for over 400 proteins from more than 20 major pathways in E. coli metabolism. Complementing these methods, we constructed a series of synthetic genes to produce concatenated peptides (QconCAT) for absolute quantification of the proteins and made them available through the Addgene plasmid repository (vvww.addgene.org). To facilitate high sample throughput, we developed a fast, analytical flow chromatography method using a 5.5 mm gradient (10 min total run time). Overall this toolkit provides an invaluable resource for metabolic engineering by increasing sample throughput, minimizing development time and providing peptide standards for absolute quantification of E. cob proteins. Ks. (C) 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.