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

Abstract

Transformation of engineered Escherichia coli 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 (www.addgene.org). To facilitate high sample throughput, we developed a fast, analytical-flow chromatography method using a 5.5-min 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. coli proteins.

Ten years of QconCATs: Application of multiplexed quantification to small medically relevant proteomes

“…In E. coli, QconCAT was used for the absolute quantification of over 400 proteins from more than 20 major pathways in E. coli metabolism [17]. A study on Schistosoma mansoni carried out by Castro-Borges et al to evaluate amounts of previously identified tegument proteins using the QconCAT technique reported that aquaporin is the most abundant trans-membrane protein [18].…”

Characterizing Strain Variation in Engineered E. coli Using a Multi-Omics-Based Workflow

“…Samples were analyzed using an AB Sciex (Foster City, CA) 5500Q-Trap mass spectrometer operating in MRM (SRM) mode coupled to an Agilent 1100 system. For method details, please see references (George et al, 2014) and (Batth et al, 2014). …”

Designing biomedical proteomics experiments: state-of-the-art and future perspectives

“…Furthermore miniaturization of the LC system also influences the accuracy of several important LC characteristics including control of flowrate, solvent mixing and gradient formation. For this reason, often higher flowrates are chosen for targeted proteomic applications in which high reproducibility is required [55]. In addition higher flowrates have the advantage that ultrafast gradients (UPLC) can be used increasing sample throughput.…”