title: 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.
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA 94608, USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Tanveer S
authors.initials: TS
authors.lastname: Batth
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA 94608, USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Pragya
authors.initials: P
authors.lastname: Singh
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA 94608, USA; Department of Bioengineering, University of California, Berkeley, CA 94720, USA.
authors.forename: Vikram R
authors.initials: VR
authors.lastname: Ramakrishnan
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA 94608, USA; Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology (NTNU), N-7489 Trondheim, Norway; The Proteomics and Metabolomics Core Facility (PROMEC) at NTNU, Trondheim, Norway; The Central Norway Regional Health Authority, Trondheim, Norway.
authors.forename: Mirta M L
authors.initials: MML
authors.lastname: Sousa
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA 94608, USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Leanne Jade G
authors.initials: LJG
authors.lastname: Chan
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA 94608, USA; Sandia National Laboratories, Livermore, CA 94550, USA.
authors.forename: Huu M
authors.initials: HM
authors.lastname: Tran
authors.affiliation: Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Eric G
authors.initials: EG
authors.lastname: Luning
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA 94608, USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Eva H Y
authors.initials: EHY
authors.lastname: Pan
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA 94608, USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Khanh M
authors.initials: KM
authors.lastname: Vuu
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA 94608, USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Department of Bioengineering, University of California, Berkeley, CA 94720, USA; Department of Chemical & Biomolecular Engineering, University of California, Berkeley, CA 94720, USA.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
authors.affiliation: Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Department of Bioengineering, University of California, Berkeley, CA 94720, USA.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Adams
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA 94608, USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. Electronic address: cjpetzold@lbl.gov.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Petzold
mesh_terms: D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D025941:Protein Interaction Mapping / Q000379:methods; D040901:Proteomics / Q000379:methods
chemical_list: D029968:Escherichia coli Proteins; D010455:Peptides
keywords: Absolute protein quantification; E. coli; High-throughput proteomics; Peptide standards; Targeted proteomics