Nonreducing two-dimensional polyacrylamide gel electrophoretic analysis of human colonic proteins.
Immunochemical detection of proteins with antigenic determinants that are dependent on the native spatial conformation of the protein can often pose problems with conventional two-dimensional polyacrylamide gel electrophoresis (2-DE). For example, many antigenic determinants are readily destroyed by reducing agents and/or urea, reagents which are critical components of many of the conventional isoelectric focusing and immobilized-pH-gradient (IPG) protocols used in the first electrophoretic dimension. Here we describe the use of commercially available precast 2-DE gels for performing nonreducing/non-urea 2-DE of proteins extracted from the human colon cancer cell line LIM 1215 with 0.3% Triton X-100 that permit the identification of antigens with conformational determinants by immunoblot analysis. Previous, related studies demonstrated the usefulness of peptide-mass fingerprinting for identifying 2-DE resolved proteins. Here we show how partial protein sequence data obtained by rapid peptide mapping, using capillary column liquid chromatography directly coupled with electrospray ionization tandem mass spectrometric methodologies, enhances the usefulness of this approach for identifying incompletely resolved proteins. The nonreducing 2-DE gel images reported in this study, along with our master 2-DE gel protein database for both normal human colonic crypts and several colon-cancer-derived cell lines, and information regarding microtechniques employed in this laboratory for obtaining structural data on 2-DE resolved proteins can be accessed over the Internet using World Wide Web (URL address: http:@www.ludwig.edu.au).
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003106:Colon / Q000737:chemistry*; D003195:Computer Communication Networks; D015180:Electrophoresis, Gel, Two-Dimensional; D005782:Gels; D006801:Humans; D007525:Isoelectric Focusing / Q000295:instrumentation; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011506:Proteins / Q000302:isolation & purification*; D014407:Tumor Cells, Cultured
D005782:Gels; D011506:Proteins