The purpose of this project is to develop the analytical technology required for the elution and microsequencing of proteins from two-dimensional polyacrylamide gels. Initial work has been focused on the sequence analysis of proteins that are selectively expressed during the course of multistage hepatocellular carcinogenesis. The availability of such a technique will facilitate the construction of DNA probes such that both gene cloning and studies on gene expression can be achieved. Microscale procedures for the isolation of proteins from polyacrylamide gels have been examined. Although electroelution and formic acid extraction from the gel into solution gave a high recovery of protein, sequence analysis and amino acid analysis was greatly complicated by contamination with gel components and N-terminal blocking of the proteins during isolation, such that microquantities of proteins could not be analyzed using these techniques. Electroblotting techniques were investigated for the transfer of protein to a solid matrix prior to direct gas phase sequencing. As little as 15 pmole of protein has been directly transferred from a gel to derivatized glass fiber paper using this technique. In addition the N-terminal sequence analysis of 29 pmole protein directly applied to derivatized glass fiber paper has been accomplished. Work is currently underway to optimize conditions to allow the direct sequence analysis of microquantities (less than 20 pmole) of transblotted protein. In addition methodology for the amino acid sequencing of N-terminally blocked proteins is under development.
