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

Tanveer S Batth; Pragya Singh; Vikram R Ramakrishnan; Mirta M L Sousa; Leanne Jade G Chan; Huu M Tran; Eric G Luning; Eva H Y Pan; Khanh M Vuu; Jay D Keasling; Paul D Adams; Christopher J Petzold

doi:10.1016/j.ymben.2014.08.004

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

Tanveer S Batth, Pragya Singh, Vikram R Ramakrishnan, Mirta M L Sousa, Leanne Jade G Chan, Huu M Tran, Eric G Luning, Eva H Y Pan, Khanh M Vuu, Jay D Keasling, Paul D Adams, Christopher J Petzold

31 Citations (Scopus)

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.

Original language	English
Journal	Metabolic Engineering
Volume	26
Pages (from-to)	48-56
Number of pages	9
ISSN	1096-7176
DOIs	https://doi.org/10.1016/j.ymben.2014.08.004
Publication status	Published - 1 Nov 2014

Keywords

Escherichia coli/metabolism
Escherichia coli Proteins/metabolism
Gene Expression Profiling/methods
High-Throughput Screening Assays/methods
Peptides/genetics
Protein Engineering/methods
Protein Interaction Mapping/methods
Proteomics/methods

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Batth, T. S., Singh, P., Ramakrishnan, V. R., Sousa, M. M. L., Chan, L. J. G., Tran, H. M., Luning, E. G., Pan, E. H. Y., Vuu, K. M., Keasling, J. D., Adams, P. D., & Petzold, C. J. (2014). A targeted proteomics toolkit for high-throughput absolute quantification of Escherichia coli proteins. Metabolic Engineering, 26, 48-56. https://doi.org/10.1016/j.ymben.2014.08.004

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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.",

keywords = "Escherichia coli/metabolism, Escherichia coli Proteins/metabolism, Gene Expression Profiling/methods, High-Throughput Screening Assays/methods, Peptides/genetics, Protein Engineering/methods, Protein Interaction Mapping/methods, Proteomics/methods",

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doi = "10.1016/j.ymben.2014.08.004",

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AU - Chan, Leanne Jade G

AU - Tran, Huu M

AU - Luning, Eric G

AU - Pan, Eva H Y

AU - Vuu, Khanh M

AU - Keasling, Jay D

AU - Adams, Paul D

AU - Petzold, Christopher J

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N2 - 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.

AB - 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.

KW - Escherichia coli/metabolism

KW - Escherichia coli Proteins/metabolism

KW - Gene Expression Profiling/methods

KW - High-Throughput Screening Assays/methods

KW - Peptides/genetics

KW - Protein Engineering/methods

KW - Protein Interaction Mapping/methods

KW - Proteomics/methods

U2 - 10.1016/j.ymben.2014.08.004

DO - 10.1016/j.ymben.2014.08.004

M3 - Journal article

C2 - 25205128

SN - 1096-7176

VL - 26

SP - 48

EP - 56

JO - Metabolic Engineering

JF - Metabolic Engineering

ER -

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

Abstract

Keywords

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