Previously unidentified rat liver membrane glycoproteins, whose regulation is qualitatively and quantitatively altered during the course of chemically-induced hepatocarcinogenesis, have been observed by utilizing two dimensional poly-acrylamide gel electrophoresis (2D-PAGE). The main goal of this project is to purify and characterize the specific glycoproteins which demonstrate such differences in expression in the plasma membranes of normal and neoplastic rat livers. Previous results established the N-terminal amino acid sequence for four of nine glycoproteins purified and analyzed from a single 2D-PAGE experiment. The remaining five components of interest were not sequencable in this manner, presumably because of blocked N-termini. For this reason, research was continued to develop procedures for obtaining amino acid sequence information from all of these proteins, whether blocked or unblocked. This work focuses on methods development in areas that have become critical to obtain results from minute quantities of proteins isolated from 2D-gels. Various techniques involved in amino acid sequencing of transblotted samples have been notably improved. A new type of sequencer sample support, Immobilon-CD, has been tested for efficiency in sequence analysis and transblotting. In conjunction with various projects, about 800 amino acid residues have been identified in numerous unknown protein samples from widely different sources. Excellent progress continues to be made in sequencing proteins purified from 1D- and 2D-gels. Several peptides which can be utilized to monitor various chemical reactions used in protein chemistry have been redesigned. These peptides are now routinely utilized in numerous controls and a noninterfering internal standard is used during actual sequencing of sample unknowns. As improvements are made in various sequencing techniques, they are applied in collaborative experiments in which the unknown proteins are relatively easy to purify. Following successes with these, they will also be utilized on the internal sequencing of the liver membrane glycoproteins which are important in the mechanism of neoplastic development, as described above.
