R. Tonge et al., Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology, PROTEOMICS, 1(3), 2001, pp. 377-396
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.