Reymond MA, Sanchez JC, Hughes GJ, Gunther K, Riese J, Tortola S,
Peinado MA, Kirchner T, Hohenberger W, Hochstrasser DF, Kockerling F.
Standardized characterization of gene expression in human colorectal epithelium
by two-dimensional electrophoresis.
Electrophoresis. 1997 Dec;18(15):2842-8. doi: 10.1002/elps.1150181520.
New diagnostic and prognostic markers are needed in colorectal cancer. They can
be found by differential analysis at DNA, RNA or protein level. The accuracy of
phenotypic comparisons of tumor and normal tissues depends on the purity of the
samples. We present an effective method to identify and isolate proteins that are
differentially expressed under altered conditions, and a two-dimensional
reference protein map of the normal human colonic epithelium. Normal colonic
mucosa, primary tumors and liver metastases were prepared in the operating room.
After washing in an ice-cold medium containing protease inhibitors, crypts were
isolated by mechanical preparation without using metalloproteinases. Epithelial
cells were then selected using Ber-EP4 Dynabeads. The samples were denaturated
before processing for immobilized pH gradient two-dimensional polyacrylamide gel
electrophoresis according to SWISS-2DPAGE standards. The samples contained more
than 95% epithelial cells as confirmed by fluorescence-activated cell sorting
using pan-anticytokeratin antibodies. Cell surfaces were not damaged, as assessed
by scanning electronic microscope. A protein reference map of the normal colonic
epithelium was defined. Using gel matching, N-terminal sequencing and/or
immunoblotting techniques, 60 polypeptides - including proteins specifically
expressed in colorectal epithelium - have now been identified. This reproducible
method of sample preparation permits the comparison of protein patterns found in
various pathological states with the present reference map
(http://www.expasy.ch). Some of these patterns might provide diagnostic or
prognostic markers, or even molecular targets for therapy in the future.
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