The purpose of this project is to develop the novel analytical technology required for the elution and microsequencing of proteins from two-dimensional polyacrylamide gels. The availability of such a technique will allow the construction of DNA probes in order that both gene cloning and studies on gene expression can be achieved. This will enable studies of the regulation of gene expression at the transcription, translation and post-translation levels to be undertaken. Protein sequence information will permit the chemical synthesis of small peptides which will be used for the production of antibodies. These will greatly facilitate the large scale purification of the protein and also aid in their identification and quantitation. Initial investigations have been focused on the development of microscale procedures for sample handling, enzymic digestion and peptide purification by reverse-phase HPLC. Electroelution of proteins from polyacrylamide gels was studied using I-125 labeled proteins. As little as 15 pmoles of protein could be recovered in good yield (greater than 60%) from coomassie blue stained gels. In contrast the yield of protein from silver stained gels was poor. The feasibility of protein extration by transblotting onto nitrocellulose followed by solubilization of the nitrocellulose is currently being evaluated. Methodology for the microscale trypsin digestion of proteins, followed by HPCL of the peptide fragments, is also being developed. Preliminary studies using carbonic anhydrase as a model peotide have been successful and amino acid analysis of the tryptic peptides was performed. Work is presently underway to sequence microquantities of these peptides using gas chromatography combined with electron impact mass spectrometry.
