PMID:
     12469338
Authors:
     Yan JX, Devenish AT, Wait R, Stone T, Lewis S, Fowler S.
Title:
     Fluorescence two-dimensional difference gel electrophoresis and mass spectrometry 
     based proteomic analysis of Escherichia coli.
Journal:
     Proteomics. 2002 Dec;2(12):1682-98. doi: 
Abstract:
     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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