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 sequenceable 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 in order 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 low volume reaction cartridge for the sequencer was used and the conditions modified for our specific purposes. This resulted in the attainment of sequencing data from transblotted samples that is equal to those obtained from the direct application of purified proteins to the sequencer on a polybrene-coated glass filter. In a test case we have identified 3 of 5 proteins analyzed that were purified from whole dog serum using a single 2D-polyacrylamide gel. We are currently making excellent progress in developing procedures to obtain internal sequence data from proteins purified in this manner.
