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 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. This information will aid in understanding their role either as markers or causal agents during cell transformation. Previous results established the N-terminal amino acid sequence for 4 of 9 glycoproteins purified and analyzed from a single 2D-PAGE experiment. The remaining 5 components of interest were not sequencable in this manner, presumably because of blocked N-termini. For this reason, research was continued to improve yields of starting materials and to further develop procedures for obtaining amino acid sequence information from all of these proteins, whether blocked or unblocked. The purification of the glycoproteins was modified by utilizing the detergent CHAPS in the elution buffers instead of NP-40. This allowed for better UV monitoring during all isolation steps. A general HPLC method was developed to measure the recovery of standard proteins from various experiments involving different types of concentration steps, PAGE, transblotting, and elution procedures. A major obstacle in obtaining samples for sequencing from 2D-PAGE experiments was found to be the elution of proteins or peptides from the transblotting membranes. Numerous elution buffers were evaluated to determine which was best suited to remove the components of interest. Different types of membranes, detection methods, and transblotting conditions were compared to determine which were best suited for our particular application. The ABI gas phase sequencer was modified to increase its sensitivity severalfold.
