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

Abstract

Fluorescence two-dimensional differential gel electrophoresis (2-D DIGE*) is a new development in protein detection for two-dimensional gels. Using mouse liver homogenates (control and paracetamol (N-acetyl-p-aminophenol, APAP)-treated), we have determined the quantitative variation in the 2-D DIGE process and established statistically valid thresholds for assigning quantitative changes between samples. Thresholds were dependent on normalised spot volume, ranged from approximately 1.2 fold for large volume spots to 3.5 fold for small volume spots and were not markedly affected by the particular cyanine dye combination or by multiple operators carrying out the dye labelling reaction. To minimise the thresholds, substantial user editing was required when using ImageMaster 2D-Elite software. The difference thresholds were applied to the test system and quantitative protein differences were determined using replicate gels of pool samples and single gels from multiple individual animals (control vs treated in each gel). Throughout, the differences revealed with a particular cyanine dye combination were mirrored almost without exception when the dye combination was reversed. Both pool and individual sample analyses provided unique data to the study. The inter-animal response variability in inbred mice was approximately nine times that contributed by the 2-D DIGE process. A number of the most frequently observed protein changes resulting from APAP-treatment were identified by mass spectrometry. Several of these can be rationalised based on available data on the mechanism of APAP hepatotoxicity but others cannot, indicating that proteomics can provide further insights into the biochemical basis of APAP toxicity.

Alterations of stress related proteins in genetically altered mice revealed by two-dimensional differential in-gel electrophoresis analysis

“…The CyDyes  (Cy3 and Cy5) [6] and Immobilised DryStrips  (IPG strips) were purchased from Amersham Pharmacia Biotech (Little Chalfont, Bucks, UK). Sequencing grade modified porcine trypsin was from Promega (Madison, WI, USA).…”

“…Approximately 50 mg of each protein extract (in 4 mL) were mixed with 1 mL of Cy3 or Cy5 (0.2 mM in DMF) and incubated on ice for 30 min in the dark as per Tonge et al [6]. The reactions were stopped by addition of 1 mL of 10 mM lysine and a further incubation on ice for 10 min in the dark.…”

“…Cy3 and Cy5 labelled protein images were produced by excitation of gels at 540 6 25 nm and 620 6 30 nm respectively and emission at 590 6 35 nm and 680 6 30 nm respectively using 2920 2D-Master Imager and 2D Master software (Amersham Pharmacia Biotech) as per Tonge et al [6]. Images were analyzed automatically using the Decyder Differential In-gel Analysis (DIA) software package (Amersham Pharmacia Biotech).…”

“…Such comparisons are less than ideal since gel-to-gel variations can occur due to differences in the gels and running conditions. To overcome these problems, we have used the recently developed fluorescence differential in-gel electrophoresis (DIGE) technique described by Unlu et al [5] and developed by Amersham Pharmacia Biotech [6]. In this approach, protein samples are covalently labelled with different CyDyes  prior to electrophoresis on the same gel.…”

Fluorescence two-dimensional difference gel electrophoresis and mass spectrometry based proteomic analysis of Escherichia coli

“…In this report we describe the proteomic analysis of benzoic acid induced Escherichia coli as a model system using fluorescence 2-D difference gel electrophoresis technology (2D-DIGE) [19][20][21], the principle of which was originally described by Unlu et al [22]. The use of preelectrophoretic labelling with fluorescent dyes improves the sensitivity and dynamic range of protein detection.…”