Accuracy of two-dimensional electrophoresis for target discovery in human colorectal cancer.
Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is increasingly used for target discovery in human disease to complement genomic studies. We have assessed the possibilities and limits of 2-D PAGE applied to human colorectal cancer. Up to 10(8) epithelial cells were purified from paired normal and pathological biopsies using Ber-EP4 coated magnetic beads, allowing the elimination of cellular and fluid contaminations. The mean coefficient of variation (CVAR) of repeated 2-D PAGE analysis with silver staining was lying between 20 and 28%. However, only 47% (interrun) to 76% (intrarun) of spots could be matched within a triplicate experiment. Interindividual phenotypic variability was high. Intratumoral phenotypic variability was not found to be significant. When method and tumor variability were added, 90% of CVAR were inferior to 48%. Thus, two-fold up- or down-regulation of protein expression reveals biological significance. Serial paired comparison of 923 proteins in 10 patients showed highly reproducible differences between normal and cancer tissues. Under well defined experimental conditions and after the high variability of the technique has been considered, 2-D PAGE parallel analysis of paired colorectal samples allows patient-specific tumor profiling.
Ott
V
V
Department of Surgery, University of Geneva, Switzerland.
Guenther
K
K
Steinert
R
R
Tortola
S
S
Borisch
B
B
Schlegel
W
W
Reymond
M A
MA
D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D015180:Electrophoresis, Gel, Two-Dimensional; D004847:Epithelial Cells / Q000473:pathology; D006801:Humans; D007091:Image Processing, Computer-Assisted; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics*; D010641:Phenotype; D014176:Protein Biosynthesis; D011489:Protein Denaturation; D011506:Proteins / Q000235:genetics; D020543:Proteome / Q000235:genetics*; D012015:Reference Standards; D012016:Reference Values; D015203:Reproducibility of Results; D016622:Silver Staining; D013048:Specimen Handling
D009363:Neoplasm Proteins; D011506:Proteins; D020543:Proteome