Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology

Fluorescence two-dimensional differential gel electrophoresis (2-D DIGE) is a new development in protein detection for two-dimensional gels. Using mou se liver homogenates (control and paracetamol (N-acetyl-p-aminophenol, APAP )-treated), we have determined the quantitative variation in the 2-D DIGE p rocess and established statistically valid thresholds for assigning quantit ative changes between samples. Thresholds were dependent on normalised spot volume, ranged from approximately 1.2 fold for large volume spots to 3.5 f old for small volume spots and were not markedly affected by the particular cyanine dye combination or by multiple operators carrying out the dye labe lling reaction. To minimise the thresholds, substantial user editing was re quired when using imageMaster (TM) 2D-Elite software. The difference thresh olds were applied to the test system and quantitative protein differences w ere determined using replicate gels of pool samples and single gels from mu ltiple individual animals (control vs treated in each gel). Throughout, the differences revealed with a particular cyanine dye combination were mirror ed almost without exception when the dye combination was reversed. Both poo l and individual sample analyses provided unique data to the study. The int er-animal response variability in inbred mice was approximately nine times that contributed by the 2-D DIGE process. A number of the most frequently o bserved protein changes resulting from APAP-treatment were identified by ma ss spectrometry. Several of these can be rationalised based on available da ta on the mechanism of APAP hepatotoxicity but others cannot, indicating th at proteomics can provide further insights into the biochemical basis of AP AP toxicity.