Biochemical and Biophysical Research Communications, Jun 1, 2006
FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts ribofla... more FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl(2), as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8+/-1.3nmol of FAD synthesized/min/mg protein and exhibited a K(M) value for FMN of 1.5+/-0.3microM. This is the first report on characterization of human FADS, and the first cloning and over-expression of FADS from an organism higher than yeast.
Mass spectrometry (MS) is a sensitive and powerful analytical technique, in which ionized sample ... more Mass spectrometry (MS) is a sensitive and powerful analytical technique, in which ionized sample molecules are separated according to their mass to charge ratios (m/z) by the application of electric and/or magnetic fields. If the ionization regime deposits sufficient excess energy, a proportion of the sample molecules will dissociate, the pattern of product ions formed being dependent on the structure of the intact compound (Fig. 1). A mass spectrum thus consists of the masses (strictly mass to charge ratios, m/z) of these ions plotted against abundance. Interpretation of the spectrum thus affords information about both the mol wt and the structure of the sample. By the standards of most other physical methods, mass spectrometry is fairly sensitive, requiring somewhere between low picomoles and nanomoles of material, depending on the ionization method employed, but against this must be set its destructive nature. The present introduction aims to provide a brief overview of the technique, to define some of the key terms, and to offer a short tour of some of the different instruments that are more or less legitimately called mass spectrometers. Readers wishing a more detailed account should consult refs. 1-9. Arecent volume of Methods in Enzymology (5) devoted entirely to mass spectrometry is particularly recommended, since both instrumentation and applications are comprehensively covered.
Separation and relative quantitation of complex protein mixtures remain two of the most challengi... more 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.
Biochemical and Biophysical Research Communications, Jun 1, 2006
FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts ribofla... more FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl(2), as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8+/-1.3nmol of FAD synthesized/min/mg protein and exhibited a K(M) value for FMN of 1.5+/-0.3microM. This is the first report on characterization of human FADS, and the first cloning and over-expression of FADS from an organism higher than yeast.
Mass spectrometry (MS) is a sensitive and powerful analytical technique, in which ionized sample ... more Mass spectrometry (MS) is a sensitive and powerful analytical technique, in which ionized sample molecules are separated according to their mass to charge ratios (m/z) by the application of electric and/or magnetic fields. If the ionization regime deposits sufficient excess energy, a proportion of the sample molecules will dissociate, the pattern of product ions formed being dependent on the structure of the intact compound (Fig. 1). A mass spectrum thus consists of the masses (strictly mass to charge ratios, m/z) of these ions plotted against abundance. Interpretation of the spectrum thus affords information about both the mol wt and the structure of the sample. By the standards of most other physical methods, mass spectrometry is fairly sensitive, requiring somewhere between low picomoles and nanomoles of material, depending on the ionization method employed, but against this must be set its destructive nature. The present introduction aims to provide a brief overview of the technique, to define some of the key terms, and to offer a short tour of some of the different instruments that are more or less legitimately called mass spectrometers. Readers wishing a more detailed account should consult refs. 1-9. Arecent volume of Methods in Enzymology (5) devoted entirely to mass spectrometry is particularly recommended, since both instrumentation and applications are comprehensively covered.
Separation and relative quantitation of complex protein mixtures remain two of the most challengi... more 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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