The main objective of this project is to isolate, purify, and structurally characterize the specific glycoproteins whose expression is qualitatively and quantitatively altered during chemically induced hepatocarcinogenesis in order to understand their biological functions during cell transformation. A major aspect of this work has become the development methodology for obtaining internal amimo acid sequence information on these glycoproteins isolated from 2D-gels. 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 has been utilized and the programs optimized for our specific purposes. This resulted in obtaining sequencing data from HPLC purified peptides which is equal to that observed for proteins using a polybrene-coated glass filter. In test cases, we have identified several proteins from widely different sources that were purified using 1D or 2D-polyacrylamide gels. We continue to make excellent progress in sequencing proteins purified in this manner. We have also synthesized several peptides which can be utilized to monitor various chemical reactions used in protein chemistry. We are patenting the peptide which can be utilized as a non-interfering internal standard during actual sequencing of sample unknowns. We are now applying these developed techniques, in collaborative experiments, to unknown proteins that are relatively easy to purify. Following successes with these, we will again attempt internal sequencing of the liver membrane glycoproteins which are important in the mechanism of neoplastic development, as mentioned above.
