| Metadata |
| datasetIdentifier | PASS00273 |
| datasetType | MSMS |
| submitter | Myriam Ferro <myriam.ferro@cea.fr> |
| submitter_organization | |
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| datasetTag | ecoli_identif |
| datasetTitle | Mass spectrometry-based workflow for accurate quantification of E. coli enzymes: how proteomics can play a key role in metabolic engineering |
| publicReleaseDate | 2013-12-20 00:00:00 |
| finalizedDate | 2013-07-19 02:43:53 |
| summary | Metabolic engineering aims to design high performance microbial strains producing compounds of interest. This requires systems-level understanding; genome-scale models have therefore been developed to predict metabolic fluxes. However, multi-omics data including genomics, transcriptomics, fluxomics and proteomics may be required to model the metabolism of potential cell factories.
Recent technological advances to quantitative proteomics have made mass spectrometry-based quantitative assays an interesting alternative to more traditional immuno-affinity based approaches. This has improved specificity and multiplexing capabilities. In this study we developed a quantification workflow to analyse enzymes involved in central metabolism in Escherichia coli (E. coli). This workflow combined full-length isotopically labelled standards with Selected Reaction Monitoring (SRM) analysis. First, full-length 15N labelled standards were produced and calibrated to ensure accurate measurements. Liquid chromatography conditions were then optimised for reproducibility and multiplexing capabilities over a single 30-minute LC-MS analysis. This workflow was used to accurately quantify 22 enzymes involved in E. coli central metabolism in a wild-type reference strain and two derived strains, optimised for higher NADPH production. In combination with measurements of metabolic fluxes, proteomics data can be used to assess different levels of regulation, in particular enzyme abundance and activity. This provides information which can be used to design specific strains used in biotechnology. In addition, accurate measurement of absolute enzyme concentrations is key to the development of predictive kinetic models in the context of metabolic engineering.
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| contributors | Mathieu Trauchessec , Michel Jaquinod, Aline Bonvalot, Virginie Brun, Christophe Bruley, Delphine Ropers, Hidde de Jong, Jérôme Garin, Gwenaëlle Bestel-Corre, Myriam Ferro |
| publication | unpublished |
| growth | Cultures were performed in minimal media containing 5 g/L glucose
Cells were harvested during the exponential growth phase, based on OD at 600 nm. |
| treatment | |
| extraction | Cultures were centrifuged 5 min at 8000 g, supernatant was discarded, and cells were washed in the same volume of fresh PBS. Cells were resuspended in 100 mM KPO4, pH 7.6 buffer for sonication. Lysates were centrifuged 10 min at 12,000 g and supernatant was filtered through 0.22 µm membranes. |
| separation | |
| digestion | Proteins or E. coli lysates were solubilised in 8 M urea, 50 mM NH4HC03 buffer, reduced for 30 min at room temperature with 10 mM DTT and alkylated for 45 min at room temperature in the dark with 100 mM iodoacetamide. Samples were diluted with 50 mM NH4HC03 to reach a final urea concentration of 2 M. LysC digestion was performed for 2 hours at 37 °C at an enzyme/protein ratio of 1/100 (w/w). Samples were diluted once again with 50 mM NH4HC03 to reduce urea to 0.5 M final concentration before performing trypsin digestion for 2 hours at 37 °C at an enzyme/protein ratio of 1/20 (w/w). Peptide samples were finally desalted on Sep-Pack tC18 1cc vacuum cartridges. |
| acquisition | MS/MS analyses were performed on a hybrid triple quadrupole/ion trap mass spectrometer (4000 QTRAP; ABSciex, Les Ulis, France). Liquid chromatography (LC) separation was performed on an ultimate 3000 LC-chromatography system (Dionex, Voisins le Bretonneux, France) coupled to a Kinetex XB-C18 column, 2.1 x 150 mm, 2.6 µm, 100Å (Phenomenex, Torrance, CA 90501). Peptides were separated using a linear 4% to 45% acetonitrile gradient over 30 min at a flow rate of 50 µL/min.
For MS/MS experiments, peptides were analysed on a 4000 QTRAP hybrid triple quadrupole mass spectrometer (ABSciex, Les Ulis, France) operated using a Turbo V source in Electrospray Ionisation (ESI) mode, operated in "Information Dependent Acquisition" (IDA) mode under Analyst version 1.5.1 software (AB SCIEX). A precursor ion scan between m/z 400-1400 was performed as a survey scan for the IDA method. Enhanced product ion (EPI) spectra were acquired with a scan speed of 4000 amu/s using a dynamic fill time for optimal MS/MS and rolling collision energy settings. Precursor and product ion accuracies were 1.2 and 0.6 Da, respectively, which is in line with the specifications of the 4000 QTRAP.
Data were acquired in positive mode with the ion spray voltage at 5500 V, curtain gas 15 (arbitrary units), interface heater temperature 350 °C. Collision exit, entrance and declustering potential were set to 27, 12 and 50 volts, respectively. |
| informatics | MS/MS spectra were assigned to peptides using a sequence database search strategy performed using the Ecoli_K12_decoy protein sequence databank (release July 28, 2011, 25096 sequences) and Mascot (version 2.4). Trypsin was used to generate peptides allowing 1 miss-cleavage; cystein carbamidomethyl was set as a fixed modification while mono and di-oxidation of methionine was set as a variable modification. Mass tolerance for precursors and fragments ions was 1.2 and 0.6 Da respectively. No contaminants were excluded. Peptide charge was set at 1, 2 and 3 and instrument was ESI-TRAP. |
| instruments | QTRAP 4000 |
| species | E. coli |
| massModifications | C+57.021464 |
