Database: PubMedEntry: 12469338
Original site: 12469338
Yan JX, Devenish AT, Wait R, Stone T, Lewis S, Fowler S.
Fluorescence two-dimensional difference gel electrophoresis and mass spectrometry
based proteomic analysis of Escherichia coli.
Proteomics. 2002 Dec;2(12):1682-98. doi:
Separation and relative quantitation of complex protein mixtures remain two of
the most challenging aspects of proteomics. Here an advanced technique called
fluorescence difference 2-D gel electrophoresis technology (2D-DIGE) has been
applied to a model system study of the Escherichia coli proteome after benzoic
acid treatment. The molecular weight and charge matched cyanine dyes enable
pre-electrophoretic labelling of control and treated samples which are then mixed
and run in the same gel. Pooled control and treated samples labelled with Cy
trade mark 3 were used as an internal standard for both Cy5 labelled control and
treated E. coli samples. Together with DeCyder trade mark imaging analysis
software, more accurate quantitative analysis than conventional two-dimensional
polyacrylamide gel electrophoresis was achieved. Using matrix-assisted laser
desorption/ionization-time of flight and quadrupole-time of flight mass
spectrometry a total of 179 differentially expressed protein spots were
identified. These included enzymes, stress related and substrate (e.g. amino
acids, maltose, ribose and TRP repressor) binding proteins. Of the spots
analysed, 77% contained only one protein species per spot, hence the change in
protein expression measured was solely attributed to the identified protein. Many
membrane proteins and protein isoforms were identified indicating both adequate
solubilization of E. coli samples and potential post-translational modification.
The results indicate that the regulatory mechanisms following benzoic acid
treatment of E. coli are far more complicated than hitherto expected.
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