2-DIMENSIONAL ELECTROPHORETIC ANALYSIS OF PROTEINS EXPRESSED BY NORMAL AND CANCEROUS HUMAN CRYPTS - APPLICATION OF MASS-SPECTROMETRY TO PEPTIDE-MASS FINGERPRINTING

Protein patterns of normal human colonic crypts, isolated from differe nt regions of the large intestine, and several colorectal cancer cell lines were compared using two-dimensional electrophoresis gels (2-DE). As detected by intrinsic radiolabeling and Coomassie Brilliant Blue s taining, the protein patterns for normal crypts isolated from the asce nding, and descending, regions of the colon and the rectum, were almos t (> 95 %) identical. While 75-80% of the protein spots from normal cr ypts and the colorectal cancer cell line (LIM 1863), a cell line that grows as organoids and differentiates spontaneously into crypt-like st ructures in vitro, can be matched, the relative expression levels of a large number of proteins differ. At least two protein spots (undetect able in the protein pattern from normal cells), proteins a (M(r) simil ar to 18 000, pI 6.7-6.9) and b (M(r) similar to 24 000, pI 5.9-6.0), were detected in the 2-DE gel protein pattern in the three cell lines LIM 1863, LIM 1215 and LIM 1899. The identity of these proteins is not yet known and further studies are required before they can be conside red as potential colon tumor markers. Approximately 60% of the cellula r proteins from LIM 1215 cells, a colon carcinoma cell line that exhib its many properties associated with columnar cells, can be matched wit h LIM 1863 cells. The results presented here represent an initial phas e in our efforts to develop a comprehensive protein database for norma l human colon cells and several colorectal cancer cell lines. While ou r initial protein identification relied on microsequencing methodologi es, we are presently evaluating peptide-mass fingerprinting, utilizing capillary reversed-phase high-performance liquid chromatography (RP-H PLC) and electrospray mass spectrometry, as a means for rapid identifi cation of proteins at subpicomole levels. Using this approach, protein #3 (M(r) similar to 66 000, pI 6.2) was identified as heat shock prot ein 60 from as few as seven tryptic peptide masses when they were scre ened against the molecular weight search (MOWSE) peptide-mass database .