The reproducibility of conventional two-dimensional (2D) gel electrophoresis can be improved using differential in-gel electrophoresis (DIGE), a new emerging technology for proteomic analysis. In DIGE, two pools of
proteins are labeled with 1-(5-carboxypentyl)-1'-propylindocarbocyanine halide (Cy3) N-hydroxy-succinimidyl
ester and 1-(5-carboxypentyl)-1'-methylindodi-carbocyanine halide (
Cy5) N-hydroxysuccinimidyl
ester fluorescent dyes, respectively. The labeled
proteins are mixed and separated in the same 2D gel. 2D DIGE was applied to quantify the differences in
protein expression between
laser capture microdissection-procured esophageal
carcinoma cells and normal epithelial cells and to define
cancer-specific and normal-specific
protein markers. Analysis of the 2D images from
protein lysates of approximately 250,000
cancer cells and normal cells identified 1038
protein spots in
cancer cell lysates and 1088
protein spots in normal cell lysates. Of the detected
proteins, 58 spots were up-regulated by >3-fold and 107 were down-regulated by >3-fold in
cancer cells. In addition to previously identified down-regulated
protein annexin I,
tumor rejection antigen (gp96) was found up-regulated in esophageal
squamous cell cancer. Global quantification of
protein expression between
laser capture-microdissected patient-matched
cancer cells and normal cells using 2D DIGE in combination with mass spectrometry is a powerful tool for the molecular characterization of
cancer progression and identification of
cancer-specific
protein markers.